Adaptive optics system and optical device

ABSTRACT

The present invention is intended to provide an adaptive optics system and an optical device that allow correction of wavefront phase aberration with higher accuracy than before and have a wider correction range than the conventional ones, regardless of the distance between the observation target and the fluctuation layer, and the size of the observation target. An adaptive optics system includes: a wavefront phase modulator that makes aberration correction to incident light and emits the corrected light; and an imaging-conjugated position adjustment mechanism that adjusts freely within a specimen the position of a surface imaging-conjugated with a fluctuation correction surface formed by the wavefront phase modulator. The imaging-conjugated position adjustment mechanism adjusts the fluctuation correction surface to be imaging-conjugated with a fluctuation layer existing in the specimen.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of copending U.S. application Ser.No. 15/023,281 filed Mar. 18, 2016, which is the National Phase under 35U.S.C. § 371 of International Application No. PCT/JP2014/074837, filedon Sep. 19, 2014, which claims the benefit under 35 U.S.C. § 119(a) toPatent Application No. 2013-195943, filed in Japan on Sep. 20, 2013, allof which are hereby expressly incorporated by reference into the presentapplication.

TECHNICAL FIELD

The present invention relates to an adaptive optics system and anoptical device including the adaptive optics system, more specifically,to a technique for correcting aberration resulting from an observationtarget.

BACKGROUND

Microscopic devices are generally used to observe biological specimenssuch as cells. When the observation target is a cell, however, therearises a problem that the cell surface or a specific subcellularorganelle forms a fluctuation (distortion) generating layer to causewave aberration. In addition, when the observation target is abiological tissue or organ, the tissue surface or a specific tissuelayer forms a main distortion generating layer. Accordingly, variousstudies have been conventionally conducted for microscopic devices foruse in observation of biological specimens to correct wave aberrationresulting from the observation target and obtain high-qualitymicroscopic images (refer to Patent Documents 1 to 4).

For example, Patent Document 1 proposes a technique for correctingaberration by which a rear pupil in an optical system is segmented andeach segment is controlled by a wavefront modulation device. PatentDocument 2 proposes a method for wavefront correction using an opticalwriting-type liquid crystal spatial phase modulation element. At awavefront correction imaging device described in Patent Document 2,light from an object to be measured is passed through a disturbancemedium in the space between the object and an observation surface andentered into a phase modulation surface of the liquid crystal spatialphase modulation element, and an interference pattern reflecting a phasedistribution of the disturbance medium is obtained from reference lightreflected on the phase modulation surface, and the interference patternis applied to a writing surface of the liquid crystal spatial phasemodulation element to form a phase modulation surface in such a manneras to cancel out the phase distribution of the disturbance medium, andthen light to be measured having passed from the object through thedisturbance medium and entered into the phase modulation surface andreflected on the same is observed.

Further, in the field of ophthalmic equipment, there are proposedadaptive optics systems that correct wavefront aberration detected by awavefront sensor with the use of a wavefront corrector such as adeformable mirror or a spatial light modulator as described in PatentDocuments 3 and 4.

CITATION LIST Patent Literatures

[Patent Document 1] JP-T No. 2012-533069

[Patent Document 2] JP-A No. 2002-040368

[Patent Document 3] JP-T No. 2005-501587

[Patent Document 4] JP-A No. 2011-239884

SUMMARY OF INVENTION Technical Problem

However, according to the conventional adaptive optics systems describedabove, it is difficult to correct wavefront phase aberration with highaccuracy when the observation target and the fluctuation layer are closeto each other or when the observation target is minute. In particular,biological tissues including cells have large and dense fluctuations inmany cases. As a result, the correction control is likely to becomeunstable and the range of effective correction is narrow.

It has been noted that the adaptive optics system described in PatentDocument 4 is complicated in device configuration. To reduce the devicesize, it is proposed to use a special optical system using asphericlenses or special components such as a light-driven modulator. In thatcase, however, the adaptive optics system loses simplicity andflexibility and becomes deteriorated in productivity as practicalequipment and extensibility as an experimental device.

A major object of the present invention is to provide an adaptive opticssystem and an optical device that allow correction of wavefront phaseaberration with higher accuracy than before and have a wider correctionrange than the conventional ones regardless of the distance between theobservation target and the fluctuation layer and the size of theobservation target.

Solution to Problem

An adaptive optics system according to the present invention includes: awavefront phase modulator that makes aberration correction to incidentlight and emits the corrected light; and an imaging-conjugated positionadjustment mechanism that adjusts freely within a specimen the positionof a surface imaging-conjugated with a fluctuation correction surfaceformed by the wavefront phase modulator, and the imaging-conjugatedposition adjustment mechanism adjusts the fluctuation correction surfaceto be imaging-conjugated with a fluctuation layer existing in thespecimen.

In the adaptive optics system, as the imaging-conjugated positionadjustment mechanism, an objective lens, and a first lens and a secondlens constituting relay lenses may be arranged sequentially from thespecimen side between the wavefront phase modulator and the specimen.

In this configuration, the position of the surface imaging-conjugatedwith the fluctuation correction surface in the specimen can be adjustedby changing the optical distance between the objective lens and thefirst lens.

In that case, a turn-back optical system including at least one mirrormay be arranged between the objective lens and the first lens, forexample, so that the turn-back optical system can be moved in adirection parallel to an optical axis to change the optical distancebetween the objective lens and the first lens.

In addition to the foregoing configuration or aside from the foregoingconfiguration, the position of the surface imaging-conjugated with thefluctuation correction surface in the specimen may be adjusted bychanging the optical distance between the second lens and the wavefrontphase modulator.

In that case, a turn-back optical system including at least one mirrormay be arranged between the second lens and the wavefront phasemodulator, for example, so that the turn-back optical system can bemoved in the direction parallel to an optical axis to change the opticaldistance between the second lens and the wavefront phase modulator.

Alternatively, a turn-back optical system including at least one mirrormay be arranged between the first lens and the second lens, so that theturn-back optical system can be moved in the direction parallel to anoptical axis to change the optical distance between the first lens andthe second lens.

The turn-back optical system may be placed on a slide stage movable inthe direction parallel to the optical axis.

Alternatively, the objective lens may be movable integrally with a stageon which the specimen is placed and the first and second lenses may bemovable.

The adaptive optics system of the present invention may further have awavefront sensor that detects a wavefront residual component included inthe light corrected by the wavefront phase modulator and a first controlunit that controls the wavefront modulator based on the results ofdetection by the wavefront sensor, and the first control unit may adjustthe wavefront modulator such that the fluctuation correction surface isphase-conjugated with the fluctuation layer existing in the specimen.

In that case, the first control unit can adjust the wavefront phasemodulator such that the wavefront phase of incident light on thewavefront sensor takes a set value.

At least one of the first lens and the second lens may be displaced tocorrect a wavefront tilt and/or a wavefront curvature.

A plurality of wavefront phase modulators may be arranged to beimaging-conjugated onto different positions of the specimen in a depthdirection between the specimen and the wavefront sensor.

A field stop may be arranged on or around a focal plane between thewavefront phase modulator and the wavefront sensor.

In that case, the field stop can be moved according to the position of areference object existing in the specimen.

The wavefront sensor may be changed in position according to theposition of the reference object existing in the specimen.

A plurality of wavefront sensors may be provided.

The wavefront sensor may be arranged such that the alignment of theelements is rotated 45° relative to the wavefront phase modulator.

Alternatively, the wavefront sensors may be of a phase contrast type.

An optical device according to the present invention includes theadaptive optics system described above.

The optical device of the present invention has an imaging element thatacquires an image of an observation target in the specimen and an imageof the fluctuation correction surface, and adjusts the focuses of theimages formed on the imaging element to acquire one of the image of theobservation target and the image of the fluctuation correction surface.

Alternatively, the optical device may have a first imaging element thattakes an image of an observation target in the specimen, a secondimaging element that takes an image of the fluctuation correctionsurface, and one or more beam splitters that branch part of the lightfrom the specimen toward the first imaging element and the secondimaging element, and the optical device may be configured to acquireindependently the image of the observation target and the image of thefluctuation correction surface.

In that case, the optical device may have a second control unit thatcontrols position adjustment of the surface to be imaging-conjugatedwith the fluctuation correction surface by the imaging-conjugatedposition adjustment mechanism based on the image of the fluctuationcorrection surface.

The optical device may acquire a group of tomographic images of thespecimen while shifting the focus in the depth direction at specificintervals.

The optical device may continuously acquire the image of the observationtarget in the specimen at certain time intervals.

The optical device of the present invention is a microscopic device, atelescope, a laser measurement device, a laser injection device, acamera, or a medical testing device, for example.

The microscopic device is any of a fluorescence microscope, adifferential interference microscope, a phase-contrast microscope, asuper-resolution microscope, a scanning microscope, a multiphotonmicroscope, and a laser injection microscope, for example.

Advantageous Effects of Invention

According to the present invention, the fluctuation correction surfaceand the fluctuation layer are imaging-conjugated with each other in theadaptive optics system, and it is thus possible to correct wavefrontphase aberration with high accuracy in a wide range even when theobservation target and the fluctuation layer are close to each other orwhen the observation target is minute.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram illustrating a configuration of amicroscopic device in a first embodiment of the present invention;

FIG. 2 is a schematic diagram illustrating a configuration example of aspecimen 1;

FIG. 3 is a diagram illustrating a configuration of a scaling relayoptical system holding both imaging relationship in a 4f optical systemand the planarity of wavefront phase;

FIG. 4 is a diagram illustrating a configuration example of an adaptiveoptics system in the microscopic device illustrated in FIG. 1;

FIGS. 5A and 5B are schematic diagrams illustrating operations of animaging-conjugated position adjustment mechanism for fluctuationcorrection surface, and FIG. 5A illustrates the state before adjustmentand FIG. 5B illustrates the state after adjustment;

FIGS. 6A, 6B and 6C are diagram showing a method for adjusting an imagefocus by adjustment of the adaptive optics system;

FIG. 7 is a diagram illustrating a method for an image focus in themicroscopic device via the adaptive optics system;

FIG. 8 is a diagram illustrating a configuration example of an adaptiveoptics system in a microscopic device as a first modification example ofthe first embodiment of the present invention;

FIGS. 9A and 9B are schematic diagrams illustrating operations of animaging-conjugated position adjustment mechanism for fluctuationcorrection surface in the adaptive optics system illustrated in FIG. 8,and FIG. 9A illustrates the state before adjustment and FIG. 9Billustrates the state after adjustment;

FIG. 10 is a diagram illustrating a configuration example of an adaptiveoptics system in a microscopic device as a second modification exampleof the first embodiment of the present invention;

FIG. 11 is a diagram showing a method for changing independently a lightpath length lc₂ by adjustment of a light path length lr;

FIG. 12 is a diagram showing a method for adjusting light path lengthsin a microscopic device as a third modification example of the firstembodiment of the present invention;

FIG. 13 is a diagram illustrating a specific configuration example ofimplementing the method for adjusting a light path length shown in FIG.12;

FIG. 14 is a diagram illustrating a specific configuration example ofimplementing the method for adjusting a light path length shown in FIG.12;

FIG. 15 is a diagram illustrating a specific configuration example ofimplementing the method for adjusting a light path length shown in FIG.12;

FIG. 16 is a diagram illustrating a specific configuration example ofimplementing the method for adjusting a light path length shown in FIG.12;

FIG. 17 is a diagram illustrating a specific configuration example ofimplementing the method for adjusting a light path length shown in FIG.12;

FIG. 18 is a diagram illustrating an imaging-conjugated positionadjustment mechanism (with one relay lens) for fluctuation correctionsurface in a microscopic device as a fourth modification example of thefirst embodiment of the present invention;

FIG. 19 is a diagram illustrating another imaging-conjugated positionadjustment mechanism (with two relay lenses) for fluctuation correctionsurface in the microscopic device as the fourth modification example ofthe first embodiment of the present invention;

FIG. 20 is a diagram illustrating a specific configuration example ofthe imaging-conjugated position adjustment mechanism for the fluctuationcorrection surface illustrated in FIG. 18;

FIGS. 21A and 21B are diagrams illustrating configuration examples of anadaptive optics system in a microscopic device as a fifth modificationexample of the first embodiment of the present invention;

FIG. 22 is a diagram illustrating a configuration example of animaging-conjugated position adjustment mechanism for fluctuationcorrection surface in a microscopic device as a sixth modificationexample of the first embodiment of the present invention;

FIG. 23 is a diagram illustrating another configuration example of animaging-conjugated position adjustment mechanism for fluctuationcorrection surface in the microscopic device as the sixth modificationexample of the first embodiment of the present invention;

FIG. 24 is a diagram illustrating an overview of a microscopic device asa seventh modification example of the first embodiment of the presentinvention;

FIG. 25 is a diagram illustrating a configuration of the adaptive opticssystem illustrated in FIG. 24 using a plurality of wavefront sensors;

FIG. 26 is a diagram illustrating a method for correcting a wavefronttilt component with displacement of lenses in a relay optical system;

FIG. 27 is a diagram illustrating a method for correcting a wavefrontcurvature component with displacement of the lenses in the relay opticalsystem;

FIG. 28 is a diagram illustrating a configuration example of an adaptiveoptics system in a microscopic device as a tenth modification example ofthe first embodiment of the present invention;

FIG. 29 is a schematic diagram illustrating the element layout of awavefront phase modulator and a wavefront shape in a waffle mode;

FIGS. 30A and 30B are diagrams illustrating arrangements of elements ofa wavefront sensor relative to the wavefront phase modulator, and FIG.30A illustrates a normal arrangement and FIG. 30B illustrates a 45°rotated arrangement;

FIGS. 31A and 31B are diagrams illustrating the relationship betweendifferences in element arrangement of wavefront sensor and detectionsensitivity in the waffle mode, and FIG. 31A illustrates a normalarrangement and FIG. 31B illustrates a 45° rotated arrangement;

FIGS. 32A and 32B are diagrams illustrating the relationship between the45° rotated arrangement of the wavefront sensor and the changes inmagnification ratio of the optical system, and FIG. 32A illustrates thestate before application and FIG. 32B illustrates the state afterapplication;

FIG. 33 is a schematic diagram illustrating a configuration of a laserinjection microscope using a laser injection device according to a thirdembodiment of the present invention;

FIG. 34 is a schematic diagram illustrating a configuration of aphase-contrast microscopic device according to a fourth embodiment ofthe present invention;

FIG. 35 is a schematic diagram illustrating a configuration of adifferential interference microscopic device according to a fifthembodiment of the present invention;

FIG. 36 is a schematic diagram illustrating a configuration of aconfocal scanning microscopic device according to a sixth embodiment ofthe present invention;

FIG. 37 is a schematic diagram illustrating a configuration of amultiphoton excitation microscope according to a seventh embodiment ofthe present invention;

FIG. 38 is a schematic diagram illustrating a configuration of atelescopic device according to a ninth embodiment of the presentinvention;

FIG. 39A is a photomicrograph of an artificial specimen taken such thatwavefront correction is made by a conventional adaptive optics systemwithout imaging-conjugated position adjustment, and FIG. 39B is aphotomicrograph of an artificial specimen taken such that wavefrontcorrection is made while a fluctuation compensation surface isimaging-conjugated with a fluctuation layer by the adaptive opticssystem of the present invention;

FIGS. 40A and 40B are conceptual diagrams illustrating principles ofexpansion of a viewing area; and

FIG. 41A is a photomicrograph of onion epidermal cells corrected by aconventional adaptive optics system without imaging-conjugated positionadjustment, and FIG. 41B is a photomicrograph of onion epidermal cellsin which a fluctuation correction surface is corrected to beimaging-conjugated with a fluctuation layer by the adaptive opticssystem of the present invention.

DESCRIPTION OF EMBODIMENTS

Description of embodiments of the present invention will be describedbelow in detail with reference to the accompanying drawings. However,the present invention is not limited to the embodiments described below.

First Embodiment

First, a microscopic device according to a first embodiment of thepresent invention will be described, taking a fluorescence microscope asan example. FIG. 1 is a schematic diagram illustrating a configurationof the microscopic device of the embodiment, and FIG. 2 is a schematicdiagram illustrating a configuration example of a specimen 1.

[Entire Configuration]

The microscopic device of the embodiment includes an adaptive opticssystem so that the position of an imaging-conjugated surface relative toa fluctuation correction surface of the adaptive optics system is freelyadjustable. Specifically, as shown in FIG. 1, the microscopic device ofthe embodiment includes a light source 3, a wavefront phase modulator 6,a wavefront sensor 7, an imaging camera 8, a pupil camera 9, a computer10, and others.

In the microscopic device, an objective lens Lo, a beam splitter BS1,mirrors M1 and M2, a relay lens L1, mirrors M3 and M4, and a relay lensL2 are arranged in this order between the specimen 1 and the wavefrontphase modulator 6. In addition, a beam splitter BS2, a filter F3, arelay lens L3, a field stop ST, a relay lens L4, and a beam splitter BS3are arranged in this order between the wavefront phase modulator 6 andthe wavefront sensor 7.

The microscopic device of the embodiment is configured such thatexcitation light emitted from the light source 3 is applied to thespecimen 1 via a filter F1, the beam splitter BS1, and the objectivelens Lo. In addition, the microscopic device of the embodiment isconfigured such that the light reflected on the beam splitter BS2 entersinto the imaging camera 8 via the filter F2 and the lens L5, and thelight reflected on the beam splitter BS3 enters into the pupil camera 9via the lens L6. Further, the specimen 1 and the mirrors M1 to M4 arearranged on a specimen stage 2 and slide stages 4 and 5, respectively,and are adjustable in position by moving these stages 2, 4, and 5.

[Specimen 1]

The specimen 1 observed by the microscopic device of the embodiment is abiological specimen such as an animal tissue, a plant tissue, or acultured cell that is placed on a slide glass (not illustrated) andsealed with a cover glass 103 as illustrated in FIG. 2, for example. Inthe case of the specimen 1 illustrated in FIG. 2, excitation lightemitted from the light source 3 enters into the specimen 1 via anobjective lens 104 and the cover glass 103.

The specimen 1 includes an observation target 100, a reference object101, and fluctuation elements. The “observation target” here refers to aportion (matter) that exists within the specimen 1 and is to be observedfrom its optical image, such as a biological tissue, a cell, anintracellular structure, or a molecule of fluorescent protein, forexample. The “reference object” refers to an object for use inmeasurement of wavefront fluctuations at the time of control of theadaptive optics system, and may be artificial or natural matter, such asa fluorescent bead, a tissue, a specific site in a cell, or a moleculeof fluorescent protein, for example. The observation target 100 may beused as the reference object 101. In that case, light from theobservation target 100 is introduced into the adaptive optics system forwavefront correction.

The “fluctuation elements” refer to factors that cause phase disturbanceto light from the observation target 100 or the reference object 101 atthe time of passage and fluctuates the transmitted wavefront, such asunevenness in refractive index inside the specimen 1, asperities in thesurface of the specimen 1, and the like. The fluctuations in thetransmitted wavefront constitute a cause of image deterioration. Thereis a fluctuation layer 102 including a large number of fluctuationelements resulting in error such as phase aberration between theobservation target 100 and the objective lens 104. Specific examples ofthe fluctuation layer 102 are the surface of a biological tissue ororgan, the surface of a cell (a boundary with water or culture medium),and an intracellular structure and tissue significantly different inrefractive index from the circumference, such as a cell wall of a plantcell and a chloroplast, for example.

[Specimen Stage 2]

The specimen stage 2 displaces the position of the specimen 1 alongthree axes of x, y, and z (three directions) relative to the objectivelens. At the microscopic device of the embodiment, the specimen stage 2is displaced in the z-axis direction to adjust focus, and the specimenstage 2 is displaced in the x- and y-axis directions to adjust theposition of the observation target 100 within a field of view.

[Light Source 3]

The light source 3 is intended to apply excitation light for generationof fluorescent light to the specimen 1, and may be a halogen lamp, atungsten lamp, a mercury lamp, an LED (light emitting diode), a solidstate plasma light source, various lasers, or the like.

[Filter F1]

The light source filter F1 lets through only the light with wavelengthsnecessary for excitation of fluorescent light out of the excitationlight emitted from the light source 3, and shuts off the light withunnecessary wavelengths.

[Beam Splitter BS1]

The beam splitter BS1 is a fluorescence excitation mirror that entersthe excitation light emitted from the light source 3 into the specimen 1via the objective lens 104 (objective lens Lo) when fluorescent light isto be excited in the observation target 100 and/or the reference object101 of the specimen 1. For example, in the configuration illustrated inFIG. 1, the beam splitter BS1 reflects the excitation light with shortwavelengths, and lets through the fluorescent light with longwavelengths emitted from the observation target 100 or the referenceobject 101 and guides the same to the observation-side optical system(mirror M1). The beam splitter BS1 may be a half mirror such as adichroic mirror, for example, to discriminate reflection andtransmission depending on light wavelengths.

[Objective Lens Lo]

The objective lens Lo may be an objective lens for infinity focusoptical system, for example. The objective lens for infinity focusoptical system converts divergent light from the observation target 100placed within an operating distance of the objective lens 104 intoparallel light beams as illustrated in FIG. 2, and is designed to havesmaller optical aberration in that case.

[Conjugate Slide]

The two mirrors M1 and M2 are arranged at a 90° angle on a conjugateslide stage 4. The light emitted from the specimen 1 is folded back bythe mirrors M1 and M2 in the direction parallel to the incidentdirection. The conjugate slide stage 4 is movable along the optical axesof the incident light and the outgoing light, thereby achieving variableadjustment of a light path length from the relay lens L1 to theobjective lens Lo. At the microscopic device of the embodiment, thelight path length is changed in this manner to adjust theimaging-conjugated position relative to the fluctuation correctionsurface in the specimen 1. The “light path length” here refers to thelength of a space along the light beam, and indicates the length of theoptical axis of the light flux or the optical distance between theoptical elements in the microscopic device of the embodiment.

[Focus Slide]

The two mirrors M3 and M4 are arranged at a 90° angle on the focus slidestage 5. The light reflected on the mirror M2 and entered into themirrors M3 and M4 via the relay lens L1 is folded back by the mirrors M3and M4 in the direction parallel to the incident direction. The focusslide stage 5 is also movable along the optical axes of the incidentlight and the outgoing light, thereby achieving variable adjustment of alight path length. To adjust the focus slide stage 5, the wavefrontphase modulator 6 to relay lens L2 are arranged in proper positions, thelight path length from the relay lens L1 to the relay lens L2 isadjusted, and the focus slide stage 5 is moved such that the image focusbecomes correct.

[Relay Lenses L1 and L2]

The relay lenses L1 and L2 constitute a relay optical system usingoptical elements with positive refractive power such as convex lenses.The major functions of the relay lenses L1 and L2 are as follows:

(1) Determining the image magnification ratio in conjunction with theobjective lens Lo.

(2) When projecting the light beam from the pupil opening of theobjective lens Lo onto the wavefront phase modulator 6, adjusting thelight flux to match the opening of the wavefront phase modulator 6.

(3) Making imaging conjugate between the incident surface and theoutgoing surface, and preventing unnecessary elements such as excessivetilt or curvature from increasing in wavefront phase between theincident surface and the outgoing surface.

To realize the functions (1) and (2), a scaling optical system may beused as the relay optical system. In addition, the 4f optical system isknown as a relay optical system realizing the function (3). Accordingly,the microscopic device of the embodiment also supports the case wherethe focal distances of the two relay lenses L1 and L2 are differentbased on the configuration of the 4f optical system to achieve the imagescaling ratio. Specifically, the scaling relay lens system asillustrated in FIG. 3 is formed by the relay lenses L1 and L2, a lens110 with a refractive power of 1/f1, and a lens 111 with a refractivepower of 1/f2.

In this case, first, as illustrated by dotted lines in FIG. 3, points onan incident surface P_(1n) and an outgoing surface P_(out) are in theimaging-conjugate relationship. Meanwhile, as illustrated by solid linesin FIG. 3, parallel light beams on the incident surface P_(1n) are keptin parallel even on the outgoing surface P_(out). Wavefront phasevertical to these light beams hold planarity and have no increase ordecrease due to tilt or curvature. When the lenses 110 and 111 are equalin refractive power as a similar property for holding the wavefrontphase in an optical system, the optical system is known as 4f Fouriertransform optical system.

[Wavefront Phase Modulator 6]

The wavefront phase modulator 6 is a device that makes dynamicaberration correction to varying phase error in incident light and emitsthe corrected light. In the adaptive optics system of the microscopicdevice of the embodiment, the wavefront phase modulator 6 makes phasecorrection. In this case, the element surface of the wavefront phasemodulator 6 serves as a fluctuation correction surface in the adaptiveoptics system.

For example, a deformable mirror formed by a micromachine driving athin-film mirror by electrostatic force may be used as the wavefrontphase modulator 6. In that case, the deformable mirror is connected to acontrol calculator (computer 10) via a digital-analog converter suchthat drive voltage is applied to the elements of a 12×12 square array,for example, based on a control signal output from a control port of thecalculator.

When the deformable mirror applies the drive voltage to each of theelements to drive individually the plurality of electrostatic elements,the thin-film mirror surface for reflection of the incident light beamis pushed and pulled, and the shape of the mirror surface deforms. Thisdisplaces a light phase distribution as needed, and allows dynamicaberration correction to the varying phase error. Instead of thedeformable mirror described above, a spatial light phase modulator usingliquid crystal or the like can be used as the wavefront phase modulator6.

[Beam Splitter BS2]

The beam splitter BS2 is a kind of half mirror that is highly efficientand flattened to prevent deterioration in performance of the opticalsystem. To split light by wavelength to improve the sensitivity, adichroic mirror may be used as the beam splitter BS2. For improvement oflight efficiency, instead of branching by the half mirror, a non-lightpermeable reflection mirror may be brought in and out to switch betweenthe light paths of reflection and transmission.

The light beams split by the beam splitter BS2 are branched into theoptical system for the imaging camera 8 and the optical system for thewavefront sensor 7. In the following descriptions, the light pathbranched by the beam splitter BS2 and having the imaging camera 8 willbe referred to as “imaging observation light path,” and the light pathbranched by the beam splitter BS2 and having the wavefront sensor 7 as“wavefront measurement light path.” The arrangements of the “imagingobservation light path” and the “wavefront measurement light path” maybe exchanged. Even in that case, the same functions and effects can beobtained.

[Filter F2]

The filter F2 is a fluorescent light filter that lets through only thelight with wavelengths necessary for observation from the observationtarget 100 out of the fluorescent light emitted from the specimen 1, andshuts off the unnecessary components.

[Lens L5]

The lens L5 is an image-forming lens that forms an image of theobservation target 100 within the specimen 1 on the imaging surface ofthe imaging camera 8.

[Imaging Camera 8]

The imaging camera 8 acquires an image of the observation target 100 andmay be a CCD (charge-coupled device) camera or the like, for example.The image on the imaging surface of the imaging camera 8 is convertedinto an electric signal and output to an image storage unit 11 of thecomputer 10.

[Filter F3]

The filter F3 is a fluorescent light filter that lets through only thefluorescent components from the reference object 101 out of the lightemitted from the specimen 1, and shuts off the unnecessary components.

[Relay Lenses L3 and L4]

The relay lenses L3 and L4 are lenses with positive refractive powersuch as convex lenses that form an imaging-conjugate between thewavefront phase modulator 6 as an incident surface and the opening ofthe wavefront sensor 7 as an outgoing surface. The relay lenses L3 andL4 are preferably arranged in the scaling relay optical system based onthe 4f optical system as illustrated in FIG. 3, such that there is nodifference in wavefront curvature between the outgoing light from thewavefront phase modulator 6 and the incident light on the wavefrontsensor 7.

[Field Stop ST]

The field stop ST lets through only the light passing through theopening hole out of the light emitted from the reference object 101, andshuts off other unnecessary light. The size of the opening hole of thefield stop ST is adjustable by a throttle mechanism. Even for unfocusedlight, the field stop ST has the effect of reducing the amount oftransmitted light by a mismatch between the opening hole and the beamlight collection. The field stop ST is arranged on or around a focalplane between the relay lenses L3 and L4. When the scaling relay opticalsystem illustrated in FIG. 3 is used for the relay lenses L3 and L4, forexample, the field stop ST is preferably arranged at the positioncorresponding to a Fourier diffractive plane DP or the position on thefront or back side of the same.

[Beam Splitter BS3]

The beam splitter BS3 is a kind of half mirror that branches part of thelight entered into the wavefront measurement light path toward the pupilcamera 9 in front of the wavefront sensor 7.

[Wavefront Sensor 7]

The wavefront sensor 7 detects a wavefront residual component notcorrected by the wavefront phase modulator 6 but left on the lightwavefront that has been emitted from the reference object 101 in thespecimen 1 and has passed through the fluctuation layer 102 and receivedfluctuations. There is no particular limitation on the type of thewavefront sensor 7. For example, the wavefront sensor 7 may be aShack-Hartmann type. The Shack-Hartmann-type wavefront sensor has alenslet array at the incident opening portion, and further includes alight-receiving CCD camera to detect the tilt of an incident wavefrontby lateral displacements of a light-collection spot of each smallopening generated at the back side of the lenslet array.

The adaptive optics system in the microscopic device of the embodimentcan control the phase tilt of incident light on the wavefront sensor 7to take an arbitrary set value, specifically, an ideal value, zero, forexample. When the wavefront sensor 7 is a Shack-Hartmann-type wavefrontsensor, the opening surface of the lenslet array constitutes thewavefront measurement surface in the adaptive optics system.

[Computer 10]

The computer 10 is provided with the image storage unit 11 that storesimages acquired by the imaging camera 8 and the pupil camera 9 and anadaptive optical control unit 12 that controls the wavefront phasemodulator 6 and others based on a signal from the wavefront sensor 7.The adaptive optical control unit 12 converts a measurement signal ofwavefront residual output from the wavefront sensor 7 into a controlvoltage correction value of the wavefront phase modulator 6 by anadaptive optical control program, and outputs the resultant value to thewavefront phase modulator 6 to compensate for a wavefront phase error onthe wavefront correction surface.

The microscopic device of the embodiment performs a closed-loop controlamong the incident wavefront, the wavefront sensor 7, the wavefrontphase modulator 6, the adaptive optical control unit 12 of the computer10 to provide negative feedback to the incident wavefront fluctuations,so that the phase distortion on the light wavefront convergesasymptotically on an ideal value, zero, for example.

[Lens L6]

The lens L6 is an image-forming lens for the pupil camera 9, which formson the imaging surface of the pupil camera 9 a mirror image 13 of thefluctuation measurement surface of the wavefront sensor 7 from the lightbranched by the beam splitter BS3 as illustrated in FIG. 1. The lens L6may be made changeable in position to form an image of the referenceobject 101 on the imaging surface of the pupil camera 9 by adjusting thefocal distance of the beam splitter BS3 at infinity (moving to theposition of a lens L6′).

[Pupil Camera 9]

The pupil camera 9 acquires an image of the fluctuation measurementsurface of the wavefront sensor 7 and an image of the reference object101. The pupil camera 9 may be a CMOS (complementary metal oxidesemiconductor) camera or the like, for example. The image generated onthe imaging surface of the pupil camera 9 is converted into anelectrical signal and output to the image storage unit 11 of thecomputer 10.

[Operations]

Next, operations of the microscopic device of the embodiment will bedescribed taking the observation of the specimen 1 illustrated in FIG. 2as an example.

<Outline of Microscopic Operations>

In the microscopic device of the embodiment, in advance, the deformablemirror is set flat and the optical components such as lenses and camerasare adjusted such that a general microscopic image can be obtainedwithout wavefront correction. Then, the specimen 1 is placed on thespecimen stage 2, the specimen stage 2 is moved while observing theobservation target 100 by the imaging camera 8, and the focus and thelateral position are adjusted such that the image becomes optimum. Theimages shot by the imaging camera 8 are stored in the computer 10. Theimages are read from the computer 10 as necessary and subjected to imageprocessing and analysis.

<Outline of Image Correction by the Adaptive Optics>

To perform image correction by adaptive optics, first, the size andposition of the field stop ST are adjusted and the specimen stage 2 ismoved to adjust the position of the specimen 1 while the image of thereference object 101 acquired by the pupil camera 9 is checked so thatlight enters appropriately into the wavefront sensor 7. The adjustmentof the field stop ST and the positioning of the specimen 1 can beautomatically performed by a position adjustment control unit (notillustrated) controlling the individual adjustment mechanisms.

In that state, the adaptive optical control unit 12 (adaptive opticalcontrol program) of the computer 10 is operated to acquire data onwavefront phase distortion at the wavefront sensor 7, provide negativefeedback of the correction value to the wavefront phase modulator 6 toupdate the control value of the mirror surface shape, and repeat theseoperations. When the feedback control is appropriately conducted, thewavefront residual detected by the wavefront sensor 7 converges on anideal value, zero, for example, and the resolution of the image on theimaging camera 8 is improved to obtain a clear image.

<Position Adjustment of the Imaging-Conjugated Surface Relative to theFluctuation Correction Surface>

FIG. 4 is a diagram illustrating a configuration example of the adaptiveoptics system in the microscopic device illustrated in FIG. 1. FIGS. 5Aand 5B includes schematic diagrams illustrating operations of animaging-conjugated position adjustment mechanism for the fluctuationcorrection surface. In FIGS. 4 and 5, dotted lines show light from theobservation target or the reference object, solid lines show lightconverging on the fluctuation correction surface, and reference signs foand f1 to f4 show the focal distances of the objective lens Lo and thelenses L1 to L4, respectively. In FIGS. 5A and 5B, reference sign P1shows the position of the observation target or the reference object,and P2 shows the position of the imaging-conjugated surface relative tothe fluctuation correction surface. FIG. 4 illustrates only the elementsrelated to the position adjustment of the imaging-conjugated surfacerelative to the fluctuation correction surface in an equivalent manner,but does not illustrate the elements not directly related to theposition adjustment.

First, descriptions will be given as to a method for adjusting theimaging-conjugated position relative to the fluctuation correctionsurface by changing the optical distance between the objective lens Loand the relay lens L1. As shown by the solid lines in FIG. 4, when thelight beam entering into the fluctuation correction surface (elementsurface) of the wavefront phase modulator 6 is reversed, the light beamconverges in the specimen 1, and an image of the fluctuation correctionsurface in the adaptive optics system is formed at a distance lc′ fromthe objective lens Lo as an imaging-conjugated surface.

As illustrated in FIG. 5A, before the position adjustment of theimaging-conjugated surface relative to the fluctuation correctionsurface, the opening pupil of the objective lens Lo and the fluctuationcorrection surface P2 are imaging-conjugated with each other, and thuslc′=0. The distance lc′ between the image-forming plane P2 of thefluctuation correction surface and the objective lens Lo can be changedby adjusting a light path length lc between the objective lens Lo andthe relay lens L1. Taking advantage of this change, the position of theimage-forming plane P2 of the fluctuation correction surface in theadaptive optics system can be imaging-conjugated with the position ofthe fluctuation layer 102 in the specimen 1.

As illustrated in FIG. 5B, after the position adjustment, the distancelc′ between the image-forming plane P2 of the fluctuation correctionsurface and the objective lens Lo is increased and the image-formingposition of the fluctuation correction surface is moved. However, thelight beam shown by the dotted lines emitted from the observation targetor the reference object remains unchanged between before and after theadjustment. Specifically, by adjusting the image-forming position of thefluctuation correction surface, the light path length lc between theobjective lens Lo and the relay lens L1 is changed but the light pathlength (=f1+f2) between the relay lens L1 and the relay lens L2 and thelight path length (=f2) between the relay lens L2 and the wavefrontphase modulator 6 remain unchanged to fix the focus of the image fromthe observation target or the reference object.

The adjustment of the light path length lc can also be made by movingthe position of the objective lens Lo together with the specimen stage 2or arranging the turn-back optical system composed of the mirrors M2 andM3 on the conjugate slide stage 4 and moving the same in the directionparallel to the optical axis. In that case, in the microscope, theconjugate slide stage 4 is operated to adjust the position of theimaging-conjugated surface relative to the fluctuation correctionsurface to the fluctuation layer 102 of the specimen 1 while checkingthe focus of the image on the fluctuation measurement surface acquiredby the pupil camera 9, changes in light and shade due to convergence anddivergence of wavefront phase, and a wavefront signal from the wavefrontsensor 7, such that the effectiveness of the adaptive optics systembecomes favorable. The conjugate slide stage 4 can be automaticallyadjusted by controlling the imaging-conjugated position adjustmentmechanism by a position adjustment control unit (not illustrated).

The image in the pupil camera 9 decreases in contrast resulting from thewavefront phase when the imaging-conjugated surface relative to thefluctuation correction surface and the fluctuation layer in the specimenbecome close to each other. Accordingly, taking advantage of this, theposition of the imaging-conjugate in the specimen relative to thefluctuation correction surface may be adjusted to be optimum for thefluctuation layer. This adjustment can also be automatically made by theposition adjustment control unit (not illustrated) controlling theimaging-conjugated position adjustment mechanism.

At this time, the light beams shown by the dotted lines from theobservation target or the reference object in FIGS. 5A and 5B areparallel between the objective lens Lo and the relay lens L1, andtherefore the adjustment made to the light path length lc has noinfluence on the focus of the observation target or the reference objectboth before and after the adjustment. Specifically, even when theconjugate slide stage 4 is moved, the state of the light passing throughthe field stop ST and the state of the light incident on the wavefrontsensor 7 illustrated in FIG. 4 are kept. In addition, when the referenceobject is placed at the same position as the observation target, thefocus and the magnification ratio of the observation target on theimaging camera 8 and the pupil camera 9 are kept. Accordingly, it ispossible to adjust freely the imaging-conjugated position in thespecimen 1 relative to the fluctuation correction surface.

By the method for adjusting the optical distance (light path length lc)between the objective lens Lo and the relay lens L1 described above, itis possible to adjust readily the position of the imaging-conjugatedsurface in the specimen 1 relative to the fluctuation correctionsurface. In addition, as in the microscopic device of the presentembodiment, by aligning the position of the imaging-conjugated surfacerelative to the fluctuation correction surface with the fluctuationlayer 102, it is possible to improve the accuracy of wavefrontcorrection and extend the viewing field of the correction region in theimage of the observation target 100 acquired by the imaging camera 8.

<Focus Adjustment>

The adaptive optics operates to sharpen the image of the referenceobject 101. Accordingly, when the reference object 101 is distant fromthe observation target 100 as illustrated in FIG. 2, for example, thereis need for a separate method for adjusting the focus on the observationtarget 100. The focus on the observation target 100 can be adjusted insuch a manner as described below.

FIGS. 6A to 6C are diagrams illustrating a method for adjusting an imagefocus by the adjustment of the adaptive optics system. In FIGS. 6A to6C, the same constituent elements as those in the microscopic device ofthe first embodiment described above are given the same reference signsas those in the first embodiment, and descriptions thereof will beomitted. FIGS. 6A to 6C illustrate only the elements related to theadjustment of the image focus, but do not illustrate the elements notdirectly related to the adjustment of the image focus.

<Focus Adjustment by the Imaging Camera 8>

Before the focus adjustment illustrated in FIG. 6A, the focus of thelight from the observation target 100 shown by broken lines is shiftedfrom the image-forming surface of the imaging camera 8. The focalposition can be adjusted by changing the focus of the lens L5 or theposition of the imaging camera 8. In this case, an aberration correctionsystem may be incorporated into the lens L5 in conjunction with thefocus adjustment.

<Focus Adjustment by Movement of the Wavefront Sensor 7 and the RelayLens>

In the wavefront correction by the adaptive optics system, elements 6 aof the wavefront phase modulator 6 are controlled such that thewavefront becomes planar on the incident surface of the wavefront sensor7, that is, the light beams incident on the wavefront sensor 7 becomeparallel. Meanwhile, as illustrated in FIG. 6B, the field stop ST, therelay lens L4, and the wavefront sensor 7 are moved separately orintegrally along the optical axis to displace the light incident on thewavefront sensor 7.

In this state, when the adaptive optics system is operated as usual tocontrol the elements 6 a of the wavefront phase modulator 6 such thatthe light incident on the wavefront sensor 7 is returned to be parallel,the light beams reflected on the wavefront phase modulator 6 can bediverged or converged. Then, taking advantage of the divergence orconvergence of the light beams caused by the wavefront phase modulator 6corresponding to the displacement of the field stop ST, the relay lensL4, and the wavefront sensor 7, it is possible to adjust arbitrarily thefocus of the light beams in the imaging observation light path or thefocus of the imaging camera 8.

<Infinity Focusing of the Imaging Observation Light Path>

By the same method, the outgoing light from the adaptive optics systemcan be subjected to infinity focusing. When the reference object 101 isdistant from the observation target 100 as illustrated in FIG. 2, thereoccurs a difference in convergence or divergence due to the focus shiftbetween the light beams shown by the broken lines from the observationtarget 100 and the light beams shown by the dotted lines from thereference object 101 as illustrated in FIG. 6A. Accordingly, when thefield stop ST, the lens L5, and the wavefront sensor 7 are displacedalong the optical axis as illustrated in FIG. 6B, the light beams shownby the dotted lines from the reference object can be entered as parallellight beams into the wavefront sensor 7, and the light beams shown bythe broken lines from the observation target can be emitted as parallellight beams to the imaging observation light path.

<Adjustment of the Curvature by Providing an Offset to the WavefrontSensor 7>

As shown in FIGS. 6A, 6B and 6C, instead of moving the positions of thelens L4 and the wavefront sensor 7 to absorb the convergence ordivergence of the light (dotted lines) from the reference object, byproviding an intentional deviation corresponding to the convergence ordivergence of the light, that is, providing an offset to the signal fromthe wavefront sensor 7, it is possible to obtain the effect of focusadjustment. At that time, it is possible to shift the focus of theoutgoing light, for example, by providing a deviation to the measurementvalue of the wavefront sensor 7, transferring the deviated value to thewavefront phase modulator 6 under negative feedback control, andadjusting the displacements of the elements 6 a caused by the deviatedvalue.

By using this method, the outgoing light to the imaging observationlight path can be adjusted to an infinity focus. In this case, asnecessary, the field stop ST is moved along the optical axis togetherwith the movement of the focus.

<Focus Adjustment and Aberration Correction>

Aberration may occur when the distance from the objective lens Lo to theobservation target is deviated from the designed value by the focusadjustment method described above, or when the state of lighttransmission through the relay lens changes. At that time, theaberration can be corrected by providing the image-forming lens L5 withan aberration correction mechanism in conjunction with the focus of thelens, adding an intentional deviation to the wavefront correction valuesof the wavefront sensor 7 and the wavefront phase modulator 6 to makefine adjustments for the aberration, or giving some contrivance to therelay lens, for example.

<Application to the Microscopic Device>

FIG. 7 is a diagram illustrating the correction of a focus shift betweenthe light (dotted lines) from the reference object and the light (brokenlines) from the observation target by the method illustrated in FIG. 6B.As illustrated in FIG. 7, the light (broken lines) from the observationtarget becomes parallel light beams, that is, an infinity focus opticalsystem at the emission from the beam splitter BS2 to the imagingobservation light path directed to the imaging camera 8. The light(broken lines) from the observation target is held parallel even fromthe relay lens L2 to the wavefront phase modulator 6. Accordingly, thedisplacement of the light path length at this section does not haveinfluence on image properties such as the focus and the magnificationratio of the image of the observation target on the imaging camera 8.

As described above in detail, in the microscopic device of theembodiment, the position of the imaging-conjugated surface relative tothe fluctuation correction surface is adjusted by the adaptive opticssystem to align with the fluctuation layer in the specimen, therebyretaining the maximum effectiveness of wavefront phase aberrationcorrection. Accordingly, it is possible to improve the correctionaccuracy as compared to the conventional ones, and achieve high-accuracycorrection even when the observation target and the fluctuation layerare close to each other or the observation target is minute. Themicroscopic device of the embodiment is high in stability even withlarge fluctuations and allows wide-range correction, as compared to theconventional systems.

Further, the microscopic device of the embodiment can reduce focus errorat the time of incidence on the wavefront sensor. This makes it possibleto suppress measurement error and enhance the effectiveness of thecorrection even in the case where the specimen has a lot of spatiallyfine structures like a biological specimen and high-spatial frequencycomponents cannot be ignored. As a result, it is possible to improve thestability of wavefront correction and achieve stable operations.

The light emitted from the observation target and the reference objectis not necessarily fluorescent light, and diverged light or reflectedlight from the observation target and the reference object may bedetected by the imaging camera 8 and the pupil camera 9. Instead of therelay lens, a reflection mirror may be used. This configuration iseffective for avoidance of color aberration in the case of usinginfrared rays.

In the microscopic device of the embodiment, the light path at thereflection side of the beam splitter BS2 and the light path at thetransmission side of the beam splitter BS2 can be exchanged to performthe same operations. Specifically, in the microscopic device illustratedin FIG. 1, although the optical system including the wavefront sensor 7is arranged in the straight transmission-side light path and the opticalsystem including the imaging camera 8 and the image-forming lens L5 isarranged in the reflection-side light path, these optical systems may beexchanged such that the optical system including the wavefront sensor 7is arranged in the reflection-side light path and the optical systemincluding the imaging camera 8 and the image-forming lens L5 is arrangedin the straight transmission-side light path.

First Modification Example of the First Embodiment

Next, a microscopic device according to a first modification example ofthe first embodiment of the present invention will be described. FIG. 8is a diagram illustrating a configuration example of an adaptive opticssystem in a microscopic device as the modification example. FIGS. 9A and9B includes schematic diagrams illustrating operations of animaging-conjugated position adjustment mechanism for fluctuationcorrection surface in the adaptive optics system. In FIG. 8, the sameconstituent elements as those of the microscopic device of the firstembodiment described above are given the same reference signs as thoseof the first embodiment, and descriptions thereof will be omitted. FIG.8 illustrates only the elements related to the position adjustment ofthe imaging-conjugated surface relative to the fluctuation correctionsurface in an equivalent manner, but does not illustrate the elementsnot directly related to the position adjustment.

The distance lc′ between the image-forming plane P2 of the fluctuationcorrection surface and the objective lens Lo can be changed not only bythe adjustment of the light path length lc₁ between the objective lensLo and the relay lens L1 illustrated in FIG. 8 but also the adjustmentof the light path length lc₂ between the relay lens L2 and the wavefrontphase modulator 6. By combination of these adjustments, theimaging-conjugated position relative to the fluctuation correctionsurface can be adjusted in a wider range.

[Configuration of the Adaptive Optics System]

In the microscopic device of the modification example, as illustrated inFIG. 8, a light path length adjustment mechanism is provided in thelight path between the relay lens L2 and the wavefront phase modulator6. There is no particular limitation on the configuration of the lightpath length adjustment mechanism. For example, as with the mirrors M1 toM4 illustrated in FIG. 1, the light path length adjustment mechanism maybe configured such that two mirrors M5 and M6 are arranged at a 90°angle on a slide stage movable along the optical axis.

[Operations]

In the microscopic device of the modification example, when the lightpath length lc₁ between the objective lens Lo and the relay lens L1 andthe light path length lc₂ between the relay lens L2 and the wavefrontphase modulator 6 become shorter, the distance lc′ between theimage-forming plane P2 of the fluctuation correction surface and theobjective lens Lo becomes longer. That is, to adjust the distance lc′between the image forming plane P2 of the fluctuation correction surfaceand the objective lens Lo, either or both of the light path length lc₁and the light path length lc₂ may be increased or decreased.

Before the position adjustment of the imaging-conjugated surfacerelative to the fluctuation correction surface, as illustrated in FIG.9A, the opening pupil of the objective lens Lo and the fluctuationcorrection surface P2 are imaging-conjugated with each other andtherefore lc′=0. Then, when the light path length lc₁ and the light pathlength lc₂ are decreased to adjust the position of the image-conjugatedsurface relative to the fluctuation correction surface, the distance lc′between the image-forming plane P2 of the fluctuation correction surfaceand the objective lens Lo increases as illustrated in FIG. 9B.

At this time, the light path length lc₁ between the objective lens Loand the relay lens L1 and the light path length lc₂ between the relaylens L2 and the wavefront phase modulator 6 are changed, but the lightpath length (=f1+f2) between the relay lens L1 and the relay lens L2remains unchanged and fixed. As illustrated in FIGS. 9A and 9B, thelight (dotted lines) from the observation target or the reference objectbecomes parallel light both before and after the position adjustmenteven between the relay lens L2 and the wavefront phase modulator 6 aswell as between the objective lens Lo and the relay lens L1.

Accordingly, the position of the image-forming plane P2 of thefluctuation correction surface is moved but the emitted light beam isunchanged between before and after the adjustment. That is, theadjustment of the light path length lc₂ has no influence on theplanarity of the light from the observation target or the referenceobject on the wavefront sensor 7. In addition, when the reference objectis placed at the same position as the observation target, the adjustmentof the light path length lc₂ also has no influence on the focus andmagnification ratio of the image of the observation target on theimaging camera 8.

In the microscopic device of the modification example, the distance lc′between the image-forming plane P2 of the fluctuation correction surfaceand the objective lens Lo is changed by the adjustment of the light pathlength lc₂. Accordingly, it is possible to improve the accuracy ofwavefront correction and expand the field of view by aligning theposition of the image-forming plane P2 of the fluctuation correctionsurface in the adaptive optics system with the fluctuation layer 102 inthe specimen 1. The configurations, operations, and effects of themodification example other than the ones described above are the same asthose of the first embodiment.

Second Modification Example of the First Embodiment

Next, a microscopic device of a second modification example of the firstembodiment in the present invention will be described. FIG. 10 is adiagram illustrating a configuration example of an adaptive opticssystem in a microscopic device of the modification example. In FIG. 10,the same constituent elements as those of the microscopic device of thefirst modification example of the first embodiment described above aregiven the same reference signs as those of the first modificationexample of the first embodiment, and descriptions thereof will beomitted. FIG. 10 illustrates only the elements related to the positionadjustment of the imaging-conjugated surface relative to the fluctuationcorrection surface in an equivalent manner, but does not illustrate theelements not directly related to the position adjustment.

When the objective lens Lo is focused on the object target at infinity,as the position of the reference object becomes distant from theobservation target, the light from the reference object becomes out offocus and no longer parallel light beams at the outgoing side of theobjective lens Lo. As a result, the light shown by the dotted lines fromthe reference object in FIG. 7 does not become parallel light beams bothbetween the objective lens Lo and the relay lens L1 and between therelay lens L2 and the wavefront phase modulator 6. Accordingly, theflexibility of adjustment of the light path length lc₁ and the lightpath length lc₂ is subjected to some constrains.

In the microscopic device of the modification example, a light pathlength lr between the relay lens L1 and the relay lens L2 is variableand adjustable arbitrarily as illustrated in FIG. 10. Accordingly, thelight (dotted lines) from the reference object becomes parallel lightbeams between the relay lens L2 and the wavefront phase modulator 6, andthe displacement of the light path length lc₂ at this section isflexible and independent from the convergence of the light beams fromthe reference object. This displacement is used for adjustment ofimaging conjugate between the fluctuation correction surface and thefluctuation layer.

[Operations]

Next, operations of the microscopic device of the modification examplewill be described. FIG. 11 is a diagram showing a method for changingindependently the light path length lc₂by adjustment of the light pathlength lr. In the microscopic device of the modification example, first,the light path length lr between the relay lens L1 and the relay lens L2is kept at the sum of focal distances (f1+f2), and the light path lengthlc₂ between the relay lens L2 and the wavefront phase modulator 6 iskept at the focal distance f2. In this state, the reference object isplaced at a focal point at the working distance of the objective lensLo, and the adaptive optics is operated. Then, the imaging-conjugatedposition between the fluctuation layer and the fluctuation correctionsurface is adjusted to look for a light path length lc₁ at which theeffectiveness of the adaptive optics becomes maximum. The decision ofthe light path length lc₁ constitutes rough adjustment of the spacingbetween the reference object and the fluctuation layer.

After that, the negative feedback control of the adaptive optics by thecontrol unit 12 of the computer 10 is temporarily stopped, and thespecimen stage 2 is adjusted to align the observation target with thefocal point at the working distance of the objective lens Lo. Then, thelight path length lr is adjusted such that the shift from the plane ofthe incident light wavefront from the reference object to the wavefrontsensor 7 becomes smallest, that is, the incident light beams become mostvertical. This adjustment can be made by the use of the image from thepupil camera 9 for checking.

Next, the adaptive optics is operated and focused on the observationtarget in that state. The method for focus adjustment is the same asthat of the first embodiment. Alternatively, in the configuration ofFIG. 10, the focus adjustment may be made by displacing the wavefrontsensor 7, the relay lens L4, and the field stop ST along the opticalaxis. In this case, the light emitted from the beam splitter BS2 to theimaging observation light path directed to the lens L5 becomes parallelbeams, that is, focused at infinity.

As illustrated in FIG. 11, in the rough adjustment of theimaging-conjugated position between the fluctuation layer and thefluctuation correction surface described above, at the stage where thelight path length lc₁ is decided, the light beams shown by the solidlines from the fluctuation layer of the specimen 1 are parallel betweenthe relay lenses L1 and L2. Accordingly, the adjustment of the lightpath length lr has no influence on the imaging conjugate between thefluctuation layer and the fluctuation correction surface. Therefore, theadjustment of the light path length lc₁ can be made with flexibility.After the adjustment of the light path length lr, the light beams fromthe reference object are parallel between the relay lens L2 and thewavefront phase modulator 6. Therefore, the adjustment of the light pathlength lc₂ can be made with flexibility, without influence on the entryof the light from the reference object into the wavefront phasemodulator 6.

As described above, in the microscopic device of the modificationexample, the light beams shown by the solid lines from the fluctuationlayer, the light beams shown by the broken lines from the observationtarget, and the light beams shown by the dotted lines from the referenceobject can be independently set in parallelism in the light path toachieve the independence of focus adjustment. Accordingly, it ispossible to reduce aberration by keeping the working distance at whichthe light beams from the observation target are incident on theobjective lens at the designed value such as an infinite distance.

Third Modification Example of the First Embodiment

Next, a microscopic device of a third modification example of the firstembodiment of the present invention will be described. The adaptiveoptics systems in the microscopic devices according to the firstembodiment and the first modification example thereof described abovecan be easily implemented. In addition, in the microscopic deviceaccording to the second modification example of the first embodiment,the fluctuation correction layer of the adaptive optics system isadjusted and imaging-conjugated with the fluctuation layer of thespecimen, and therefore the focus adjustment can be made independentlyeven when the observation target and the reference object are atdifferent positions.

However, these microscopic devices of the modification examples requirea plurality of mirrors. When a large number of mirrors are used asoptics for adjustment of the light path length in the adaptive opticssystem, the light transmission efficiency may become lower. Accordingly,the microscopic device of the modification example uses a small numberof mirrors to improve the light transmission efficiency. FIG. 12 is adiagram showing a method for adjusting light path lengths in themicroscopic device of the modification example, and FIGS. 13 to 17 arediagrams illustrating specific configuration examples. In FIGS. 12 to17, the same constituent elements as those of the first modificationexample of the first embodiment will be given the same reference signsas those of the first modification example of the first embodiment, anddescriptions thereof will be omitted.

As illustrated in FIG. 12, in the microscopic device of the modificationexample, the positions of the relay lenses L1 and L2 are variable alongthe optical axis. Accordingly, the light path length pl₁ from theobjective lens Lo to the relay lens L1, the light path length pl₂ fromthe relay lens L1 to the relay lens L2, and the light path length pl₃from the relay lens L2 to the wavefront phase modulator 6 can beseparately adjusted. Further, in the microscopic device of themodification example, a mechanism for adjustment of the entire lightpath length pl₀ from the objective lens Lo to the wavefront phasemodulator 6 is added to allow all the light path lengths to beindependently adjusted.

Moreover, by adding some contrivance to the adjustment of these lightpath lengths, it is possible to decrease the number of optical elementssuch as mirrors necessary for the adjustment of the light path lengthsand achieve the simple and high-efficiency optical system. Specificconfiguration examples for carrying out the adjustment method will bedescribed below.

[Example with the Movement of Lenses and the Slides of Turn-BackMirrors]

In the system of FIG. 13, the relay lenses L1 and L2 are movable, andthe slide stage 4 with the turn-back mirrors M1 and M2 is movable inparallel to the optical axes of the incident light and the outgoinglight. Accordingly, the light path length pl₁, the light path lengthpl₂, and the light path length pl₃ illustrated in FIG. 12 are alladjustable.

[Example with the Use of the Objective Lens and the Specimen Stage]

In the system of FIG. 14, the relay lenses L1 and L2 are movable and astage 22 on which the objective lens Lo and the specimen stage 2 areplaced is movable along the optical axis. This increases the degree offreedom of adjustment, and the light path length pl₁, the light pathlength pl₂, and the light path length pl₃ are all adjustable.

[Example with a Simplified Conjugate Adjustment Stage]

In the system of FIG. 15, the object-side focus of the objective lens Lois adjusted with the specimen 1 placed on the specimen stage 2 and mademovable. In addition, a stage 24 on which the relay lenses L1 and L2 andthe stage 4 are placed is also movable in parallel to the optical axis.Moving the stage 24 increases or decreases the light path length pl₁ andthe light path length pl₃ at the same time, thereby to increase theeffectiveness of the conjugate of the fluctuation correction surface inthe simple system. This configuration has a practical advantage in thatthe system can be simplified while providing adjustment means for theimportant light path lengths.

In this system, the stage 22 on which the objective lens Lo and thespecimen stage 2 are placed may be movable in parallel to the opticalaxis, so that the light path length pl₁ can be independently adjusted,as in the system of FIG. 14. Further, the stage 4 may be movable so thatthe light path length pl₂ can be independently adjusted.

[Example with the Use of Concave Mirrors Instead of the Relay Lenses]

In the system of FIG. 16, concave mirrors CM1 and CM2 are used insteadof the relay lenses L1 and L2 constituting the relay lenses. Theseconcave mirrors CM1 and CM2 are placed on the slide stage 4, and thestage 4 can be moved along the optical axes of the incident light andthe outgoing light. This makes it possible to increase or decrease thelight path length pl₁ and the light path length pl₃ at the same time,thereby to enhance the effectiveness of adjustment of conjugate positionof the fluctuation correction surface in the simple system.

The stage 22 on which the objective lens Lo and the specimen stage 2 areplaced and the slide stage 21 on which the concave mirror CM1 is placedare movable vertically to the moving direction of the slide stage 4.Accordingly, the light path length pl₂ can also be independentlyadjusted. In this system, the use of the mirror surface avoids coloraberration and improves the light efficiency because the convergence ofthe light and the folding of the light path are conducted at the sametime. In this system, the stage 22 on which the objective lens Lo andthe specimen stage 2 are placed may be movable in parallel to theoptical axis so that the light path length pl₁ can be independentlyadjusted.

[Example with the Use of Combined Moving and Rotational Mirrors]

In the system of FIG. 17, the positions of the relay lenses L1 and L2are variable so that the light path lengths pl₁ and pl₃ can be adjusted,and the positions and angles of the mirrors M1 and M2 are changed sothat the light path length pl₂ can be independently adjusted. Thissystem is also compatible with the case where the optical axis of theincident light from the relay lens L1 to the mirror M1 and the opticalaxis of the outgoing light from the mirror M2 to the relay lens L2 arenot parallel to each other. The same or similar functions of independentadjustment of the light path lengths can also be implemented by changingthe arrangement sequence of the relay lens L1, the mirror M1, the mirrorM2, and the relay lens L2 to another one such as the arrangementsequence of the mirror M1, the relay lens L1, the relay lens L2, and themirror M2.

Fourth Modification Example of the First Embodiment

Next, a microscopic device according to a fourth modification example ofthe first embodiment of the present invention will be described. Theimaging-conjugated position adjustment mechanism for aligning thefluctuation correction surface with the fluctuation layer can also beimplemented by using the objective lens with a finite focal distance.FIGS. 18 and 19 are diagrams illustrating an imaging-conjugated positionadjustment mechanism for the fluctuation correction surface in themicroscopic device of the modification example, and FIG. 20 is a diagramillustrating a specific configuration example.

[System with the Use of One Relay Lens]

The system with the use of one relay lens between the objective lens Loand the wavefront phase modulator 6 resembles the system in which thelight path length pl₀ is zero between the objective lens Lo and therelay lens L1 illustrated in FIG. 12. In the adjustment mechanism ofFIG. 18, the light beams shown by the dotted lines from the referenceobject and the light beams shown by the broken lines from theobservation target are made parallel between the relay lens L2 and thewavefront phase modulator 6 by the adjustment of the light path lengthpl₂.

Besides, making variable the light path length pl₃ makes it possible todisplace the position of the image on the fluctuation correction surfacegenerated in the specimen shown by the solid lines, regardless of thefocus position and the magnification ratio of the observation target orthe reference object. Accordingly, the imaging-conjugated position ofthe fluctuation correction surface relative to the fluctuation layer canbe adjusted with high flexibility. This adjustment is similar to theadjustment with the use of the objective lens focused at infinitydescribed above.

Meanwhile, as shown by the solid lines in FIG. 18, there exists a planethat converts the outgoing light from the objective lens Lo intoparallel light beams focused at infinity at the side nearer theobjective lens Lo than the observation target in the specimen. Thus, inthe microscopic device of the modification example, the wavefront phasemodulator 6 is conjugated with that plane as shown by the solid lines inFIG. 18. Accordingly, the light beams shown by the solid lines becomeparallel between the objective lens Lo and the relay lens L2, and arenot subjected to the influence of adjustment of the light path lengthpl₂. The adjustment of the light path length pl₂ between the objectivelens Lo and the relay lens L2 can be used for focus adjustment in thecase where the observation target and the reference object aredifferent, thereby realizing high-flexibility adjustment.

[System with the Use of Two Relay Lenses]

FIG. 19 illustrates a system using two relay lenses or two relay lensgroups in which the light beams shown by the solid lines from thefluctuation layer are made parallel between the relay lens L1 and therelay lens L2 by the negative or positive refractive power of the relaylens L1 and the adjustment of the light path length pl₁. Accordingly, itis possible to adjust the light path length pl₂ and adjust the planarityand focus of the light beams from the reference object or the lightbeams from the observation target, without influence on the conjugaterelationship between the fluctuation layer and the fluctuationcorrection layer.

Taking advantage of this, the light beams from the reference object orthe light beams from the observation target are adjusted to be parallelbetween the relay lens L2 and the wavefront phase modulator 6. Thismakes it possible to adjust the imaging-conjugate between thefluctuation layer and the fluctuation correction surface with highflexibility while avoiding the influence of adjustment of the light pathlength pl₃. This adjustment is similar to the adjustment with theobjective lens focused at infinity described above.

[Configuration Example of the Optical System]

The conjugate adjustment optical system using the objective lens or theobjective lens group with a finite distance focus described above canconstitute an adaptive optics system in combination with the imagingoptical system and the wavefront sensor, as in the case of the objectivelens with an infinite distance focus. For example, the system using onerelay lens illustrated in FIG. 18 is configured as illustrated in FIG.20. In the system using two relay lenses, the objective lens Lo and therelay lenses L1 and L2 illustrated in FIG. 19 may be used instead of theobjective lens Lo and the relay lens L2 illustrated in FIG. 20.

Fifth Modification Example of the First Embodiment

Next, a microscopic device of a fifth modification example of the firstembodiment of the present invention will be described. The light pathlength from the wavefront phase modulator 6 to the emission-side lensand the light path length between the wavefront sensor 7 and the lens infront of the wavefront sensor 7 can be adjusted without having to changethe focal distances of the relay lenses L3 and L4.

FIGS. 21A and 21B are diagrams illustrating configuration examples of anadaptive optics system in the microscopic device of the modificationexample. The light path length from the wavefront phase modulator 6 tothe relay lens L3 at the emission side is referred to as le, and thelight path length from the detection surface of the wavefront sensor 7to the relay lens L4 in front of the wavefront sensor 7 is referred toas lw. A method for adjusting the light path lengths le and lw will bedescribed below.

The adaptive optics system illustrated in FIG. 21A has a standardarrangement in which the light path length le is set as a focal distancef3 of the relay lens L3, and the light path length lw is set as a focaldistance f4 of the relay lens L4. Meanwhile, in the adaptive opticssystem of FIG. 21B, the light path lengths le and lw are adjusted tolight path lengths le′ and lw′, respectively. The parallel light beamcomponents shown by dotted lines in the wavefront phase modulator 6 andthe wavefront sensor 7 remain unchanged against the adjustment of thelight path lengths le and lw, and therefore there is no influence on theplanarity of the wavefront vertical to the light beams.

At the same time, the wavefront phase modulator 6 and the wavefrontsensor 7 may be imaging-conjugated with each other as shown by solidlines. In FIG. 21B, le′>f3 and lw′<f4, but the same adjustment can bemade even when le′<f3 and lw′>f4 by reversing the direction of theadjustment. Accordingly, the lengths of the light paths le and lw can beadjusted without having to change the focal distances of the relaylenses L3 and L4.

In the microscopic device of the modification example, for example, whenthe parallel light path section between the wavefront phase modulator 6and the relay lens L3 behind the wavefront phase modulator 6 is to belonger to install a light path branching mirror, filter, and the like,it is possible to set the parallel light path section in an appropriatelength without replacement of the lenses. Because of the unnecessity oflens replacement, the adjustment is easy. At the time of the adjustment,the diameter of the beams at the parallel light beam section becomesconstant at the ratio of the focal distances f3 and f4 of the lenses,and thus the magnification ratio is kept constant between the elementsurface of the wavefront phase modulator 6 and the element surface ofthe wavefront sensor 7. As described above, this adjustment method hasflexibility relative to the operations of the adaptive optics system.

Further, when the light path lengths le and lw necessary for thearrangement of equipment and components are known in advance, it ispossible to minimize the optical system by using the relay lenses L3 andL4 with as the shortest focal distances f3 and f4 as possible.

Sixth Modification Example of the First Embodiment

Next, a microscopic device according to a sixth modification example ofthe first embodiment of the present invention will be described. FIGS.22 and 23 are diagrams illustrating configuration examples of animaging-conjugated position adjustment mechanism for the fluctuationcorrection surface in the microscopic device of the modificationexample. As illustrated in FIGS. 22 and 23, the adaptive optics systemin the microscopic device of the modification example has a plurality ofwavefront phase modulators. The wavefront phase modulators areimaging-conjugated at different positions within the specimen 1.Accordingly, this configuration is compatible with the case where thereis a plurality of fluctuation layers or the fluctuation layer is thick.

In the adaptive optics system of FIG. 22, for example, two wavefrontphase modulators 16 a and 16 b are arranged on the optical path togetherwith the relay lenses L1 a, L2 a, L1 b, and L2 b, and adjusted to beimaging-conjugated at different positions in the depth direction of thespecimen 1. Accordingly, this configuration supports the correction witha plurality of fluctuation layers or a thick fluctuation layer. Adistance lca′ between the imaging-conjugated image on the fluctuationcorrection surface generated by the wavefront phase modulator 16 a andthe objective lens Lo can be changed by adjusting light path lengths lc1a and lc2 a illustrated in FIG. 22.

A distance lcb′ between the imaging-conjugated image on the fluctuationcorrection surface generated by the wavefront phase modulator 16 b andthe objective lens Lo can be adjusted by changing the light path lengthslc1 a and lc2 a and also changing light path lengths lc1 b and lc2 b. Inthe adaptive optics system, as in the case with one wavefront phasemodulator, the light beams shown by the dotted lines from the referenceobject or the observation target become parallel between the objectivelens Lo and the relay lens L1 a, between the relay lens L2 a and thewavefront phase modulator 16 a, between the wavefront phase modulator 16a and the relay lens L1 b, and between the relay lens L2 b and thewavefront phase modulator 16 b. Accordingly, it is possible to adjustfreely the distances lca′ and lcb′ between the imaging-conjugated imageson the fluctuation correction surface and the objective lens Lo. Inaddition, as in the case with one wavefront phase modulator, theoutgoing light can be focused at infinity by adjusting a light pathlength lra between the relay lens L1 a and the relay lens L2 a, and alight path length lrb between the relay lens L1 b and the relay lens L2b.

Meanwhile, as illustrated in FIG. 23, in the adaptive optics systemusing a plurality of wavefront phase modulators, no relay lens may bearranged between the wavefront phase modulators. The distance lca′between the imaging-conjugated image on the fluctuation correctionsurface generated by the wavefront phase modulator 16 a and theobjective lens Lo can be adjusted by changing the light path lengths lc1a and lc2 a. The distance lcb′ between the imaging-conjugated image onthe fluctuation correction surface generated by the wavefront phasemodulator 16 b and the objective lens Lo can be adjusted by changing thelight path lengths lc1 a and lc2 a, and also changing a light pathlength lcab.

As in the case with one wavefront phase modulator, the light beams shownby the dotted lines from the reference object or the observation targetbecome parallel on these light paths. Accordingly, the distances lca′and lcb′ between the imaging-conjugated images on the fluctuationcorrection surface and the objective lens Lo can be adjustedindependently and freely. In addition, as in the case with one wavefrontphase modulator, the outgoing light can be focused at infinity byadjusting the light path length lr between the relay lens L1 and therelay lens L2.

As for the correction of the wavefront, for example, the control unit 12of the computer 10 provides a negative feedback of the measurement valueof a wavefront residual obtained by the wavefront sensor 7 placed behindto the wavefront phase modulators 16 a and 16 b, thereby to make acorrection such that the residual decreases toward zero. At themeasurement of the wavefront residual, the optical systems are moved asnecessary such that the signal from the wavefront sensor 7 includeswavefront fluctuations on the imaging-conjugated surfaces of thewavefront phase modulators 16 a and 16 b. The optical systems moved atthat time may include the wavefront sensor 7, the relay lenses L3 andL4, and the field stop ST. From calculations based on a series ofwavefront residual measurement values, optimum control values to be fedback to the wavefront modulators 16 a and 16 b are determined and usedfor control operations.

Seventh Modification Example of the First Embodiment

Next, a microscopic device according to a seventh modification exampleof the first embodiment of the present invention will be described. FIG.24 is a diagram illustrating an overview of an adaptive optics system ina microscopic device of the modification example. FIG. 25 is a diagramillustrating a configuration of the adaptive optics system illustratedin FIG. 24 using a plurality of wavefront sensors. In the case of usinga single reference object, the field of view is limited to thecorrectable region centered around the reference object. Therefore, inthe microscopic device of the modification example, a plurality ofreference objects is used for wavefront measurement, and correctableregions are connected to expand the field of view.

As illustrated in FIG. 24, in the microscopic device of the modificationexample, the wavefront sensor 7 captures a plurality of referenceobjects RefA, RefB, and RefC at different positions, and measureswavefront fluctuation values at their corresponding effective apertures.Then, the wavefront phase modulator 6 is driven to make corrections withthese measured values as error signals. In this manner, it is possibleto expand the field of view by connecting the error signals obtained atthe respective effective apertures of the reference objects and makingcorrections to the correction region including all the effectiveapertures.

In the microscopic device of the modification example, the referenceobject can be switched by several methods such as moving the field stopST, moving the wavefront sensor 7, moving a convergence spot of theexcitation light source to shift the position where the reference objectis to be excited, subjecting the image on the wavefront sensor 7 toimage processing and cutting information on part of the referenceobject, and the like. As illustrated in FIG. 25, instead of theswitchover, a plurality of wavefront sensors 17 a to 17 c correspondingto the plurality of reference objects RefA, RefB, and RefC may beprepared and relay optical systems (lenses L4 a to L4 c and mirrors M1 ato M1 d) may be inserted between the wavefront sensors and the referenceobjects to make position adjustment relative to the reference object.

Further, by using a plurality of wavefront phase modulators, it ispossible to expand the field of view in combination with this methodeven when the fluctuation layer is thick. In this case, the wavefrontsensor 7 may be moved as necessary or a plurality of wavefront sensorsmay be used to perform wavefront measurement.

Eighth Modification Example of the First Embodiment

Next, a microscopic device according to an eighth modification exampleof the first embodiment of the present invention will be described. FIG.26 is a diagram illustrating a method for correcting a wavefront tiltcomponent with displacement of lenses in a relay optical system;

FIG. 27 is a diagram illustrating a method for correcting a wavefrontcurvature component with displacement of the lenses in the relay opticalsystem. In FIGS. 26 and 27, the dotted lines show the positions of thelight beams and the lenses when the incident wavefront has no tilt orcurvature, and the solid lines show the positions of the light beams andthe lenses when the incident wavefront has an tilt or a curvature.

[Correction of the Wavefront Tilt Component]

The wavefront tilt component can be corrected by mounting apublicly-known tip tilt mirror or a wavefront phase modulator on a tiptilt mount, for example. Otherwise, the wavefront tilt component canalso be corrected by displacing (laterally displacing) the relay lensvertically to the optical axis. For example, as illustrated in FIG. 26,when the tilt of the incident wavefront is corrected by laterallydisplacing the relay lens L1 relative to the optical axis as shown by aone-point chain line, the relay lens L2 may be further reverselydisplaced to cancel out the shake of the light beam to keep constant theposition of the outgoing light beam.

[Correction of the Wavefront Curvature Component]

The wavefront curvature component can be corrected by changing thedistance between the relay lens L1 and the relay lens L2. For example,as illustrated in FIG. 27, the relay lens L1 may be displaced along theoptical axis as shown by an one-point chain line to adjust the distancebetween the relay lens L1 and the relay lens L2.

At the corrections of the wavefront tilt and focus described above, itis possible to increase the maximum value of correctable wavefrontdistortion under a control in conjunction with the wavefront phasemodulator 6 so as to decrease the error signal from the wavefront sensor7.

Ninth Modification Example of the First Embodiment

Next, as a ninth modification example of the first embodiment of thepresent invention, the use of calculated values and the simplificationof the adjustment procedure will be described. Various set values suchas the light path length lc or the light path lengths lc1 and lc2adjusted such that the fluctuation layer and the fluctuation correctionsurface are imaging-conjugated with each other, the light path length lrdetermined by the position of the reference object, the positions of thewavefront sensor 7, the relay lens L4, and the field stop ST decidedsuch that the image comes into focus, the deviation and offset values ofthe wavefront sensor 7 and the wavefront phase modulator 6, are decidedby prescribed values of the focal distance of the optical lenses, theposition of the fluctuation layer, the position of the reference object,and others.

For these values, the set values equivalent to those obtained by theadjustment operations described above can be determined in advance bycalculations and simulations based on optical designs. Specifically, theset values may be determined in advance by the adjustment operations orthe calculations, recorded in association with the lenses to be used,the position of the reference object, and the position of thefluctuation layer, and used in the actually used system depending on thesituation of the observation. This facilitates the adjustment operationsand further allows automation of the adjustment operations.

Tenth Modification Example of the First Embodiment

Next, as a tenth modification example of the first embodiment of thepresent invention, the application to acquisition of Z stack images willbe described. In general, a group of tomographic images shot with shiftsin the focus of the microscope at specific intervals in the depthdirection of the specimen is called as Z stack images. By applying theforegoing adjustment method to the movement of the focus for acquisitionof the Z stack images, the fluctuation correction surface of theadaptive optics system and the fluctuation layer in the specimen can beimaging-conjugated with each other. Accordingly, it is possible toprevent disturbances and fluctuations at the time of the correction bythe adaptive optics.

FIG. 28 is a diagram illustrating a configuration example of an adaptiveoptics system in a microscopic device according to the modificationexample. As illustrated in FIG. 28, in the adaptive optics system in themicroscopic device of the modification example, the specimen stage 2 andthe objective lens Lo are moved integrally to adjust the light pathlengths, and the relay lenses L1 and L2 are moved along the opticalaxis, similar to the configuration of FIG. 14.

First, the focus in the specimen 1 is displaced to acquire the Z stackimages. Specifically, as illustrated in FIG. 28, the specimen stage 2 onwhich only the specimen 1 is placed is moved relative to the objectivelens Lo (displacement amount i). Subsequently, the positions of thestage 22 on which the specimen stage 2 and the objective lens Lo areplaced, and the relay lenses L1 and L2 are adjusted to displace thelight path length from the objective lens Lo to the relay lens L1, thelight path length from the relay lens L1 to the relay lens L2, and thelight path length from the relay lens L2 to the wavefront phasemodulator 6 (displacement amounts ii to iv). At that time, the positionof the fluctuation correction surface and the position of thefluctuation layer in the specimen are kept in imaging conjugate.

Next, the focus adjustment is performed by the method described abovesuch that the imaging camera 8 is imaging-conjugated with theobservation target and comes into a focus. Specifically, theimage-forming lens L5 is focus-adjusted (displacement amount v); anoffset is added to a wavefront measurement signal from the wavefrontsensor 7, a negative feedback of the offset signal is provided as adeviation signal to the elements 6 a of the wavefront phase modulator 6,and the elements 6 a are controlled and given a curvature on thereflection surface (displacement amount vi); and a slide stage 25 onwhich the field stop ST, the relay lens L4, and the wavefront sensor 7are placed is displaced and adjusted such that the incident light fromthe reference object becomes a planar wave on the wavefront sensor 7(displacement amount vii), for example.

In this manner, the displacement amounts (displacement amounts ii tovii) of the series of optical elements corresponding to the movement offocus of the objective lens on the specimen (displacement amount i) canbe determined in advance by experiments or calculations. Accordingly,these adjustments can be automatically performed during the acquisitionof the Z stack images. Similarly, while the displacement amount iillustrated in FIG. 28 is set as a fixed value, the focus of theimage-forming lens L5 can be adjusted by the displacement amount v toacquire the Z stack images. This method may increase aberration andnarrow the image focus adjustment range, but can be readily implemented.

Keeping constantly the imaging-conjugate relationship between thefluctuation layer of the specimen 1 and the fluctuation correctionsurface of the adaptive optics system makes it possible to preventdeviations of the correction values in the adaptive optics system.Accordingly, it is possible to eliminate or reduce the consumption oftime for re-correction of the adaptive optics during the acquisition ofthe Z stack images. Conventionally, there is need to operate theadaptive optics for making corrections each time when the focus isdisplaced in the Z axis direction. The application of the modificationexample can improve this situation.

Eleventh Modification Example of the First Embodiment

Next, as an eleventh modification example of the first embodiment of thepresent invention, the application to time-lapse acquiring will bedescribed. In general, the observational method for performingcontinuous acquiring at constant time intervals is called time-lapseacquiring. In this case, the time-lapse acquiring includes acquiring atlow-speed time intervals and acquiring at a video rate or higher-speedtime intervals.

Although the time-lapse acquiring is publicly known, the time-lapseacquiring in the microscopic device of the modification example isperformed while image degradation is corrected by the adaptive optics.This improves the observation accuracy. In addition, the observationaccuracy can further be increased by holding the fluctuation surfaceimaging-conjugated with the correction surface of the adaptive optics.

Specifically, once the positions of the fluctuation component and thereference object in the specimen are aligned with the objective lens Loto complete the adjustment of the imaging-conjugated position relativeto the fluctuation correction surface, even when the fluctuationcomponent changes in shape or content, the time-lapse acquiring can beperformed while the changes are corrected by the adaptive optics system.In addition, the time-lapse acquiring can be automated by the use of apublicly-known software application (Metamorph produced by MolecularDevices, LLC. or the like).

Further, by combining the automated acquisition of the Z stack imagesdescribed above with the automated time-lapse acquiring, it is possibleto acquire the Z stack images continuously to obtain 4D (3D+time)images. When the observation target is to be moved three-dimensionally,by making settings before acquiring such that the Z stack images can beacquired within the possible range of movement of the observationtarget, high-definition images of the three-dimensionally movingobservation target can be obtained while the fluctuation component iscorrected. In this manner, even when the observation target is to bemoved to change the fluctuation component in shape and content, as faras the position of the fluctuation surface is fixed, the fluctuationsurface can be adjusted to be imaging-conjugated with the correctionsurface of the adaptive optics to obtain 4D images with a furtherimprovement in observation accuracy.

Further, even when the position of the fluctuation surface is to bemoved, it is possible to control automatically the fluctuationcorrection surface and the fluctuation surface to be imaging-conjugatedwith each other based on the image acquired by the pupil camera 9.Specifically, at a stage prior to acquiring, the possible range ofmovement of the fluctuation surface is set in advance. Then, before eachtime-lapse acquiring, the Z stack images are acquired by the pupilcamera 9 within the possible range of movement of the fluctuationsurface while the distance between the objective lens Lo and the relaylens L1 or between the relay lens L2 and the wavefront phase modulator 6is changed.

When the imaging-conjugated surface relative to the fluctuationcorrection surface and the fluctuation layer in the specimen becomeclose to each other, the position adjustment control unit (notillustrated) automatically controls the imaging-conjugated positionadjustment mechanism such that the neighborhood of the fluctuation layerand the fluctuation correction surface become imaging-conjugated witheach other with a decrease in contrast resulting from the wavefrontphase of the image or the like as an index. After that, the Z stackimages are acquired by the method described above. By performing thisprocess at each time-lapse acquiring, 4D images can be obtained withimprovement in observation accuracy even when both the observationtarget and the fluctuation surface are moved.

Twelfth Modification Example of the First Embodiment

Next, as a twelfth modification example of the first embodiment of thepresent invention, a method for adjusting an excitation wavelength and afluorescence wavelength between the reference object and the observationtarget will be described. When the light beams to be detected from thereference object and the observation target are both fluorescent light,the characteristics of excitation light and fluorescence wavelength canbe shifted between the two light beams to improve distinctiveness andprevent performance degradation under mutual influences.

Specifically, the fluorescent light can be selectively excited in thereference object and the observation target by adjusting and choosingfluorescent substances for the reference object and the observationtarget with a difference in excitation wavelength characteristics, andswitching the wavelength of the light source according to theirrespective excitation wavelengths such that one excitation efficiency ishigher than the other.

In addition, fluorescent substances for the reference object and theobservation target are chosen with a difference in fluorescencewavelength characteristics, and a dichroic mirror is used as the beamsplitter BS2 as necessary to increase distinctiveness between theirrespective fluorescent light beams. Further, a wavelength filter formaking the transmission of the fluorescent light from the referenceobject higher than the transmission of the fluorescent light from theobservation object is inserted in the wavefront measurement light path,and a wavelength filter for making the transmission of the fluorescentlight from the observation target higher than the transmission of thefluorescent light from the reference object is inserted in the imagingobservation light path. This reduces mutual influences.

The distinction between the fluorescence wavelengths by the filters orthe like described above is also applicable to the case where thereference object and the observation target are excited at the same timeby a single or plural excitation light beams.

Thirteenth Modification Example of the First Embodiment

Next, as a thirteenth modification example of the first embodiment ofthe present invention, image processing using wavefront fluctuationinformation will be described. Point image distribution can be estimatedusing the information from the wavefront sensor 7 and the wavefrontphase modulator 6 in the adaptive optics system. In the microscopicdevice of the modification example, the fluctuation correction surfaceis made variable to obtain three-dimensional fluctuation information.Accordingly, it is possible to visualize the fluctuation information asan image of a three-dimensional target such as a microscopic specimen,and improve the accuracy of estimation of point image distribution. As aresult, the three-dimensional fluctuation structure and the estimationof the point image distribution can be used for image recoveryprocessing.

Second Embodiment

Next, a microscopic device according to a second embodiment of thepresent invention will be described. At present, wavefront sensors aremainly of a Shack-Hartmann type, and their adjustment principles dependon the imaging conjugate of optical surfaces. Therefore, the conjugatedposition adjustment can be made in combination with other publicly-knowntypes. The wavefront sensors may be of a curvature type, aphase-contrast type, an tilt detection type using other Hartmann maskssuch as a talbot mask. Among them, the phase-contrast type is atechnique for using the visualization and detection of optical phase bya phase-contrast method in detection of wavefront phase.

Meanwhile, when wavefront correction is made using a Shack-Hartmann-typewavefront tilt sensor or the like, some wrinkle-like deformation calledwaffle mode may be seen in the wavefront phase modulator in whichadjacent elements are alternately displaced in the vertical directionunder influence of noise. This is likely to occur at the time ofmicroscopic observation with many small fluctuations. FIG. 29 is aschematic diagram illustrating a wavefront shape in the waffle mode. Asillustrated in FIG. 29, the wavefront distortion in the waffle mode isdifficult to detect by the wavefront tilt detection method using aShack-Hartmann sensor or the like. Therefore, once the distortionoccurs, the convergence of the control becomes deteriorated.Accordingly, when a waffle-mode wavefront shape occurs under strongnoise influence, the control diverges with reduction in accuracy andbecomes unstable.

FIGS. 30A and 30B are diagrams illustrating arrangements of elements ofthe wavefront sensor relative to the wavefront phase modulator, andFIGS. 31A and 31B includes diagrams illustrating the relationshipbetween differences in arrangement of wavefront sensor elements anddetection sensitivity to the waffle mode. FIGS. 32A and 32B are diagramsillustrating the relationship between the 45° rotated arrangement of thewavefront sensor and the changes in magnification ratio of the opticalsystem. In the microscopic device of the embodiment, the arrangement ofthe elements 7 a of the wavefront sensor 7 illustrated in FIG. 30A isrotated at a 45° angle and inclined as illustrated in FIG. 30B, and theintervals between the elements 7 a are adjusted to align with thecenters of the elements 6 a adjacent in the vertical and horizontaldirections of the wavefront phase modulator 6. This makes themicroscopic device also sensitive to the waffle mode.

In the general element arrangement of FIG. 30A, each of the partiallyopening elements 7 a of the wavefront sensor 7 illustrated in FIG. 31Ais arranged at a saddle point among the four adjacent elements 6 a ofthe wavefront phase modulator 6. Therefore, the tilt resulting from thewaffle mode illustrated in FIG. 29 cannot be detected. In contrast tothis, in the 45° inclined arrangement illustrated in FIG. 30B, the tiltresulting from the waffle mode can be detected between the convex andconcave elements 6 a adjacent in the vertical and horizontal directionsof the wavefront phase modulator 6 illustrated in FIG. 31B.

Accordingly, in the microscopic device of the embodiment, to preventperformance degradation due to the waffle mode, as illustrated in FIG.32B, a Shack-Hartmann-type wavefront tilt sensor is rotated at a 45°angle around the optical axis as a rotation axis, the lengths of thefocuses of the objective lens Lo and the relay lens L1 are adjusted tochange the magnification ratio, and the elements 6 a of the wavefrontphase modulator 6 illustrated in FIG. 31A are arranged as illustrated inFIG. 31B. Accordingly, it is possible to provide sensitivity to thewaffle mode and improve the adaptive optics in operational stability.

Third Embodiment

Next, a laser injector device according to a third embodiment of thepresent invention will be described, taking the application to a laserinjector microscope as an example. The adaptive optics system describedabove can also be used to correct diffraction scattering at the time ofinjection of laser or the like into the specimen taking advantage of theregressivity of light. FIG. 33 is a schematic diagram illustrating aconfiguration of a laser injection microscope using a laser injectiondevice according to the embodiment.

As illustrated in FIG. 33, the suppression of scattering in the specimen1 can be expected by entering laser light from a laser light source LSvia the wavefront phase modulator 6 in the adaptive optics system. Afluorescent substance excitable by the incident laser can be arranged inthe reference object for operating the adaptive optics system. When theexcitable fluorescent substance is small in amount and difficult toarrange such as when using an infrared laser, fluorescent excitation bya general light source may be used as well, or an excitation laser forthe reference object different from the injection laser may be arrangedsharing the optical axis with the injection laser to excite thefluorescent substance and use the resultant fluorescent light as thereference object.

The laser injection device of the embodiment is applicable to systems inwhich the genes and substances of specific cells and cellular regionscan be optically adjusted, such as a gene induction system for specificcells using heat shock or the like (for example, an InfraRedLaser-Evoked Gene Operator manufactured by Sigmakoki Co., Ltd.) andoptogenetics.

Fourth Embodiment

Next, a phase-contrast microscopic device according to a fourthembodiment of the present invention will be described. FIG. 34 is aschematic diagram illustrating a configuration of the phase-contrastmicroscopic device according to the embodiment. A phase-contrast methodis applied to a wavefront adjusted by fluctuation correction in theadaptive optics system to improve the accuracy of optical phase imaging.

As illustrated in FIG. 34, in the phase-contrast microscope of theembodiment, a slit or pinhole PH is provided between the light source 3of a light source unit 30 and a light-collecting lens L8. Since theimage of the slit or pinhole PH of the light source unit 30 appears onthe imaging observation light path behind the beam splitter BS2, aphase-contrast mask PM is arranged at an imaging unit 80 to obtain aphase-contrast image by an image-forming lens L7 and the imaging camera8 behind the phase-contrast mask PM. In FIG. 34, the broken lines show0-order diffracted light beams, the dotted lines show the light beamsfrom the observation target and the reference object, and the solidlines show the light beams from the fluctuation correction surface.

The basic principles of phase-contrast microscopes are already publiclyknown. Incorporating the adaptive optics system into the phase-contrastmicroscope achieves a sharp phase-contrast image. In addition, adjustingthe conjugated positions of the fluctuation layer and the fluctuationcorrection surface allows the effectiveness of the adaptive optics to beachieved in a wider range and at higher accuracy. Of the correctingeffects of the adaptive optics, the detection and correction of obliquecomponents is equivalent to automatic alignment of the optical axis,thereby achieving improvement in image accuracy. This is applicable tothe automatization of alignment of the pinhole or slit by thephase-contrast method.

Fifth Embodiment

Next, a differential interference microscopic device according to afifth embodiment of the present invention will be described. FIG. 35 isa schematic diagram illustrating a configuration of the differentialinterference microscopic device according to the embodiment. By applyingdifferential interferometry to the wavefront adjusted by fluctuationcorrection in the adaptive optics system, the accuracy of optical phaseimaging can be improved as in the case of the phase-contrast methoddescribed above.

As illustrated in FIG. 35, in the differential interference microscopicdevice of the embodiment, the transmission light source 3 forillumination of the specimen 1 is dedicated for differentialinterference. A light source unit 31 lets the light from the lightsource 3 through a pinhole P, a collimator C, and a polarizing filterPL1. Then, a Wollaston polarizing prism PP1 separates the light path foreach polarization to shift the light laterally, and the condenser lensL9 projects the light onto the specimen 1. In FIG. 35, the broken linesshow 0-order diffracted light beams, the dotted lines show the lightbeams from the observation target and the reference object, and thesolid lines show the light beams from the fluctuation correctionsurface.

In the differential interference microscopic device of the embodiment,the light beams having been corrected by the adaptive optics system,entered into the light-receiving optical system on the imagingobservation light path, and separated for each polarization by theWollaston polarizing prism PP2 of the imaging unit 81, are compositedagain to cause interference. As a result, the change in the light pathlength in the spatial direction generates variations of light and shadein the image. After having passed through the polarizing prism PP2, thelight beams pass through a polarizing filter PL2, and then enter intothe imaging camera 8 through the image-forming lens L7 on the back side,thereby to obtain a differential interference image.

The basic principles of differential interference microscopic devicesare already publicly known. Incorporating the adaptive optics systeminto the differential interference microscopic device achieves a sharpdifferential interference image. In addition, adjusting the conjugatedpositions of the fluctuation layer and the fluctuation correctionsurface allows the effectiveness of the adaptive optics to be achievedin a wider range and at higher accuracy. Of the correcting effects ofthe adaptive optics, the detection and correction of oblique componentsis equivalent to automatic alignment of the optical axis, therebyachieving improvement in image accuracy.

Sixth Embodiment

Next, a confocal scanning microscopic device according to a sixthembodiment of the present invention will be described. FIG. 36 is aschematic diagram illustrating a configuration of the confocal scanningmicroscopic device according to the embodiment. It is possible toachieve performance improvement by incorporating the adjustment of theconjugated positions of the fluctuation layer and the fluctuationcorrection surface to various scanning-type adaptive opticalmicroscopes.

A specific configuration example is as illustrated in FIG. 36. Aconfocal scanning unit 82 is arranged behind the adaptive optics systemto prevent the position gap between the adaptive optics system and thespecimen 1 at the time of scanning. The scanning operation and theadaptive optical operation are independently performed to realizehigh-speed scanning. In addition, performance improvement is achieved byincorporating the adjustment of the conjugated positions of thefluctuation layer and the fluctuation correction surface. The focusadjustment may be made through the adjustment in the scanning opticalsystem or by using the focus adjustment method described above.

The confocal scanning microscopic device of the embodiment can beimplemented by arranging the confocal scanning optical system on theimaging observation light path of the beam splitter BS2. To operate theconfocal scanning microscopic device, first, the adaptive optics systemis operated to perform wavefront compensation. When the reference objectis to be excited by fluorescent light during the operation of theadaptive optics, a scanning laser may be used as far as it is on thesame wavelength.

While the pattern of the wavefront phase modulator is fixed, thespecimen 1 is scanned with laser using the confocal scanning opticalsystem arranged in the imaging observation light path of the beamsplitter BS2 to achieve high-definition confocal microscopicobservation. The basic principles of confocal microscopes are publiclyknown. By incorporating the adjustment of the conjugated positions ofthe fluctuation layer and the fluctuation correction surface into theconfocal microscope and making the adaptive optics effective in a widerrange and at higher accuracy, performance improvement can be achieved.Of the correcting effects of the adaptive optics, the detection andcorrection of oblique components is equivalent to alignment of theoptical axis, thereby achieving improvement in image accuracy byautomatic alignment of the optical axis.

Specifically, as illustrated in FIG. 36, laser light emitted from alaser light source LS passes through a polarizing element PL1 and awavelength plate WP1, then passes through a relay lens L10 and a pinholeP1, and then becomes parallel light beams through a collimate lens L7.The laser light is inclined by galvanometer mirrors GMX and GMY to scanand excite the specimen 1. The obtained fluorescent light travelsreversely from the beam splitter BS2 to the confocal scanning system 82,and is guided by the beam splitter BS3 to a photomultiplier tube PMT.

Relay lenses L11 and L12 are arranged between the beam splitter BS3 andthe photomultiplier tube PMT, and a pin hole P2 is arranged near anintermediate point between the relay lenses L11 and L12 to shut off thefluorescent light from fault planes other than the focal plane andattenuate the same. The fluorescent light passes through the pinhole P2,then passes through a wavelength plate WP2 and a polarizing element PL2,and then enters into the photomultiplier tube PMT. Accordingly, an imageformation and storage unit of a computer 14 obtains a tomographic imageof the focal plane. The configuration of the embodiment is not limitedto the scanning confocal microscope using the galvanometer mirrorsdescribed above but is also applicable to other systems with scanningmechanisms such as an optical system with a spinning disc as well as theconfocal scanning system.

Seventh Embodiment

Next, a multiphoton-excitation microscope according to a seventhembodiment of the present invention will be described. FIG. 37 is aschematic diagram illustrating a configuration of themultiphoton-excitation microscope according to the embodiment. Asillustrated in FIG. 37, the multiphoton-excitation microscope of theembodiment is provided with a laser scanning detection optical systemfor multiphoton excitation (multiphoton scanning detection unit 83) inthe imaging observation light path branched from the beam splitter BS2.

To make observations by this microscopic device, first, the adaptiveoptics system is operated to perform wavefront compensation. At thattime, when fluorescence excitation of the reference object is necessaryfor operations of the adaptive optics, two-photon excitation may becaused by a scanning laser light source LS provided in the multiphotonscanning detection unit 83. Then, the specimen is scanned by the lasertwo-photon excitation and observed by the two-photon microscope.

The basic principles of multiphoton-excitation microscopes are publiclyknown. Incorporating the adaptive optics system into themultiphoton-excitation microscope achieves high-definition two-photonmicroscopic observation. In addition, adjusting the conjugated positionof the fluctuation layer and the fluctuation correction surface allowsthe effectiveness of the adaptive optics to be achieved in a wider rangeand at higher accuracy. Of the correcting effects of the adaptiveoptics, the detection and correction of oblique components is equivalentto alignment of the optical axis, thereby achieving improvement in imageaccuracy by automatic alignment of the optical axis.

Specifically, as illustrated in FIG. 37, the laser light emitted fromthe laser light source LS passes through relay lenses L10 and L7, isinclined by the galvanometer mirrors GMX and GMY, and becomes excitationlight by multiphoton absorption to scan the specimen. The obtainedfluorescent light travels reversely from the beam splitter BS2 to themultiphoton scanning detection unit 83, and is guided by the beamsplitter BS3 to a photomultiplier tube PMT.

The relay lenses L11 and L12 are arranged between the beam splitter BS3and the photomultiplier tube PMT, and the pin hole P2 is arranged nearan intermediate point between the relay lenses L11 and L12 to shut offthe fluorescent light from fault planes other than the focal plane andattenuate the same. The fluorescent light passes through the pinhole P2,then passes through a bandpass filter BPF, and then enters into thephotomultiplier tube PMT. Accordingly, the image formation and storageunit of the computer 14 obtains a tomographic image of the focal plane.The multiphoton microscope may not have the pinhole P2, or may beconfigured such that a photoelectron detector such as a PMT is combinedwith a dichroic mirror and arranged just behind the objective lens,depending on the intended use applications such as deep-tissueacquiring. In addition, the configuration of the embodiment is notlimited to the scanning multiphoton microscope using the galvanometermirrors but is also applicable to other optical systems such as anoptical system using a spinning disc.

Eighth Embodiment

Next, a microscopic device according to an eighth embodiment of thepresent invention will be described. The adaptive optics system of thepresent invention is applicable to various microscopic devices as wellas the various microscopic devices described above. Specifically, thebasic principles of super-resolution microscopic devices are publiclyknown. Incorporating the adaptive optics system into thesuper-resolution microscopic device makes it possible to improve theconvergence performance and cross-section shapes of incoming andoutgoing wave packets and achieve performance improvement.

For example, a saturated excitation microscopic device (SAX microscope)suppresses the spread of a saturated excitation section due todegradation of light-collecting properties of incident laser resultingfrom refraction or diffraction, even under the presence of aberrationcaused by fluctuations in the observation target. In addition, the SAXmicroscope improves resolving power by correcting fluctuations indetection light entered from the specimen into the objective lens. Theimprovements in convergence performance and cross-section shapes of theincoming and outgoing wave packets by the adaptive optics can also beapplied to accuracy improvement by a method called super-resolution.

A stimulated emission depletion microscopic device (STED microscopicdevice) suppresses degradation in the shapes of excitation light spotsand stimulated emission light beams resulting from refraction anddiffraction, and corrects fluctuations in the detection light enteredfrom the specimen into the objective lens, even under the presence ofaberration caused by fluctuations in the specimen, thereby improvingresolving power.

When measuring the center of strength of the detection light positionsentered from a particle structure below diffraction limitation as aspecimen to the objective lens and the position of its center ofgravity, the STED microscopic device suppresses the degradation ofexcitation light spots resulting from refraction or diffraction evenunder the presence of aberration caused by fluctuations in the specimen,and corrects the fluctuations in the detection light entering from thespecimen into the objective lens to improve the accuracy. In addition,of the correcting effects of the adaptive optics, the detection andcorrection of oblique components is equivalent to automatic alignment ofthe optical axis, thereby achieving improvement in image accuracy.

A structured illumination microscopic device (SIM microscopic device)suppresses the degradation of illumination patterns resulting fromrefraction or diffraction even under the presence of aberration causedby fluctuations in the specimen, and corrects the fluctuations in thedetection light entering from the specimen into the objective lens toimprove the resolving power. Further, the adaptive optics system of thepresent invention is applicable to various microscopic devices such aspolarizing microscopic devices as well as the various microscopicdevices described above.

Ninth Embodiment

Next, a telescopic device according to a ninth embodiment of the presentinvention will be described. FIG. 38 is a schematic diagram illustratinga configuration of the telescopic device according to the embodiment.The technique for combining the adaptive optics with the telescopicdevice has been conventionally being studied. The technique forestablishing imaging-conjugate between the height of air fluctuationsand the fluctuation correction surface to make the adjustment permanenthas been already proposed.

In contrast to this, as in the telescopic device illustrated in FIG. 38,when the conjugated position of the adaptive optics system in thetelescopic device is freely adjustable, it is possible to adjustindependently the position of the reference object existing separatelyfrom the subject, while making adjustments such that imaging-conjugateis established between the position of fluctuations in the air or thelike in the light path to the subject and the correction surface of theadaptive optics system. This maximizes the effectiveness of the adaptiveoptics.

Further, the telescopic device of the embodiment has a system moreflexible in setting than conventional ones and thus is capable of wideapplication. Taking advantage of a wide range of adjustment function,the adaptive optics system can be designed as a general-purposereplaceable adaptive optics system, independently from object opticalsystems such as a main mirror and a sub mirror (a primary mirror and asecondary mirror) of a telescope TS. In addition, the telescopic devicemay be used as an adaptor to be combined with a camera interchangeablelens.

EXAMPLES

The effectiveness of the present invention will be specificallydescribed below, showing an example of the present invention and acomparative example. First, using the adaptive optical microscope of thepresent invention, an artificial specimen was observed through wavefrontcorrection by the conventional adaptive optics system before theimaging-conjugated position adjustment illustrated in FIG. 5A and theadaptive optics system with the fluctuation correction surfaceimaging-conjugated with the fluctuation layer illustrated in FIG. 5Baccording to the present invention, and the resultant image accuracieswere compared.

The artificial specimen was prepared by printing a grid pattern withintervals of 10 μm as an observation target on a slide glass. Thereference object was prepared by attaching 3.5 μm-diameter fluorescentbeads to the grid pattern surface. A fluctuation generation surface wasformed by attaching 50 μm-diameter glass beads as spacers to onecorrugated side of a cover glass, putting the cover glass on the slideglass such that the corrugated surface faces the slide glass, andinjecting a silicone oil with different in refractive index from theglass into the gap between the slide glass and the cover glass.

As an observation method, the adaptive optics was operated withfluorescent light from the fluorescent beads as the reference object,and then the image in a bright field was observed with focus on thegrid. FIG. 39A is a photomicrograph of the artificial specimen takensuch that wavefront correction is made by the conventional adaptiveoptics system without imaging-conjugated position adjustment, and FIG.39B is a photomicrograph of the artificial specimen taken such thatwavefront correction is made by the adaptive optics system of thepresent invention while a fluctuation correction surface isimaging-conjugated with a fluctuation layer. In FIGS. 39A and 39B, theviewing areas improved in resolution by the adaptive optics are shown bydotted lines. The fluorescent beads used as the reference object areseen at the centers of the areas shown by the dotted lines.

As compared to the image shown in FIG. 39A taken with the fluctuationcorrection surface imaging-conjugated with the pupil of the objectivelens, it can be seen that the resolution is improved in the image shownin FIG. 39B taken by a microscope using the adaptive optics system towhich the present invention is applied, by the conjugate adjustmentmechanism establishing imaging-conjugate between the fluctuation surfaceand the fluctuation correction surface.

FIGS. 40A and 40B are conceptual diagrams showing the principle ofexpansion of the viewing area. In particular, taking notice of the areaaround the field of view, light beams 201 emitted from an object 210pass obliquely through a fluctuation surface 202 and a correctionsurface 203. Accordingly, as illustrated in FIG. 40A, when thefluctuation surface 202 and the correction surface 203 do not match eachother, position error occurs due to lateral displacement of thecorrection surface resulting from the skewing of the light beams 201.The light beams 201 thus do not converge on one point but images 200 acorresponding to objects 210 a positioned at surrounding areas becomedeteriorated. As a result, it can be understood that the accuracy ofcorrection decreases at the surrounding areas and the effective range ofthe correction is limited to the central part.

In contrast, as illustrated in FIG. 40B, when the fluctuation surface202 and the correction surface 203 are adjusted and aligned with eachother according to the present invention, the fluctuation surface 202and the correction surface 203 match each other. Accordingly, noposition error occurs at the time of light transmission even when thelight beams 201 travel obliquely. As a result, the accuracy ofcorrection can be improved even when the skew of the light beamsresulting from the refraction on the fluctuation surface 202 is largeand the light beam travels obliquely in the areas surrounding the fieldof view. Accordingly, it has been confirmed that the viewing area wassignificantly expanded with higher resolution on the latter principlesthat the accuracy increases in the area surrounding the field of view toextend the correction range.

Next, epidermal cells of an onion scale leaf were observed. Fluorescentbeads were attached to the surface of the cells on the side opposite tothe objective lens, and used as a reference object. FIG. 41A is aphotomicrograph of onion epidermal cells taken such that wavefrontcorrection is made by a conventional adaptive optics system withoutimaging-conjugated position adjustment, and FIG. 41B is aphotomicrograph of onion epidermal cells taken such that wavefrontcorrection is made by the adaptive optics system of the presentinvention while a fluctuation correction surface is imaging-conjugatedwith a fluctuation layer.

The image shown in FIG. 41A was acquired with imaging-conjugate betweenthe correction surface and the opening pupil of the objective lens. Incontrast, it can be seen that, in the image shown in FIG. 41B acquiredwith the imaging-conjugate between the fluctuation correction surfaceand the fluctuation surface, the definition of the image of theintracellular tissues is improved in a field of view of about 30 μmdiameter centered around the fluorescent beads in the center of theimage used as the reference object. Accordingly, it can be understoodthat the present invention is also effective on an actual biologicalspecimen with a three-dimensional structure.

INDUSTRIAL APPLICABILITY

The adaptive optics system of the present invention is applicable tomicroscopic devices, astrometric telescopes, terrestrial telescopes,laser measurement devices, laser communication devices, underwatersurveillance cameras, positioning devices, surveying devices, energytransmission via laser, gun-sights, monitors, long-distance imagingcameras, fiber scopes in endoscopes, GRIN (GRaded INdex: gradientindex-type) fiber scopes, and other medical testing and diagnosticdevices.

The conjugated position adjustment function for fluctuation correctionlayer is expected to be effective in accuracy improvement of generalapplications of adaptive optics. The applications of the adaptive opticsinclude: the correction of fluctuations in astrometric telescopes, thecorrection of aberration in space telescopes, the stabilization of laseroscillators, the stabilization of laser optical systems, the eliminationof speckle in laser optical systems, laser nuclear fusion, plasmadensity measurement devices, beam shaping in laser processors, ocularfundus cameras, ocular fundus imaging, ocular fundus laser treatment,the correction of aberration in medical laser devices, the correction ofrefraction in a biological body by medical laser, the correction ofrefraction resulting from a biological body at testing and diagnosisusing medical devices, the correction of degradation at ground imagingfrom an artificial satellite, the correction of degradation at imagingof an artificial satellite from the ground, space optical communicationequipment, space photon communication equipment, quantum light source,quantum entangled light source, and others.

REFERENCE SIGNS LIST

-   1: Specimen-   2: Specimen stage-   3: Light source-   4, 5, 21, 22, 24, and 25: Slide stage-   6, 16 a, and 16 b: Wavefront phase modulator-   6 a: Element of wavefront phase modulator-   7, and 17 a to 17 c: Wavefront sensor-   7 a: Element of wavefront sensor-   8: Imaging camera-   9: Pupil camera-   10 and 14: Computer-   11: Image storage unit-   12: Adaptive optical control unit-   13: Wavefront sensor opening mirror image-   30 and 31: Light source unit-   80 and 81: Imaging unit-   82: Confocal scanning unit-   83: Multiphoton scanning detection unit-   100: Observation target-   101: Reference object-   102: Fluctuation layer-   103: Cover glass-   104 and Lo: Objective lens-   110, 111, L1 to L12, L1 a, L1 b, L2 a, L2 b, and L4 a to L4 c: Lens-   200: Image-   201: Light beam-   202: Fluctuation surface-   203: Correction surface-   210: Object-   BPF: Bandpass filter-   BS1 to BS3: Beam splitter-   C: Collimator-   CM1 and CM2: Concave mirror-   DP: Fourier diffraction surface-   F1 to F3: Filter-   GMX and GMY: Galvanometer mirror-   LS: Laser light source-   M1 to M6 and M1 a to M1 d: Mirror-   P, P1, and P2: Pinhole-   PH: Slit or pinhole-   PL1 and PL2: Polarizing filter-   PM: Phase-contrast mask-   PMT: Photoelectron multiplier tube-   PP1 and PP2: Wollaston polarizing prism-   ST, and STa to STc: Field stop-   TS: Telescope-   WP1 and WP2: Wavelength plate

1. An adaptive optics system comprising: a wavefront phase modulatorthat makes aberration correction to incident light and emits thecorrected light; and an imaging-conjugated position adjustment mechanismthat adjusts freely within a specimen the position of a surfaceimaging-conjugated with a fluctuation correction surface formed by thewavefront phase modulator, wherein the imaging-conjugated positionadjustment mechanism adjusts the fluctuation correction surface to beimaging-conjugated with a fluctuation layer existing in the specimen,and wherein the imaging-conjugated position adjustment mechanismincludes an objective lens and a at least one lens, the at least onelens constituting relay lens, and the objective lens and the at leastone lens are arranged sequentially in an optical path from the specimento the wavefront phase modulator.
 2. The adaptive optics systemaccording to claim 1, wherein the position of the surfaceimaging-conjugated with the fluctuation correction surface in thespecimen is adjusted by changing the optical distance between theobjective lens and the at least one lens.
 3. The adaptive optics systemaccording to claim 2, wherein a turn-back optical system including atleast one mirror is arranged between the objective lens and the at leastone lens, and the turn-back optical system is moved in a directionparallel to an optical axis to change the optical distance between theobjective lens and the at least one lens.
 4. The adaptive optics systemaccording to claim 1, wherein the position of the surfaceimaging-conjugated with the fluctuation correction surface in thespecimen is adjusted by changing the optical distance between the atleast one lens and the wavefront phase modulator.
 5. The adaptive opticssystem according to claim 4, wherein a turn-back optical systemincluding at least one mirror is arranged between the at least one lensand the wavefront phase modulator, and the turn-back optical system ismoved in the direction parallel to an optical axis to change the opticaldistance between the at least one lens and the wavefront phasemodulator.
 6. The adaptive optics system according to claim 1, whereinthe at least one lens is a first lens and a second lens, a turn-backoptical system including at least one mirror is arranged between thefirst lens and the second lens, and the turn-back optical system ismoved in the direction parallel to an optical axis to change the opticaldistance between the first lens and the second lens.
 7. The adaptiveoptics system according to claim 1, further comprising: a wavefrontsensor that detects a wavefront residual component included in the lightcorrected by the wavefront phase modulator; and a first control unitthat controls the wavefront phase modulator based on the results ofdetection by the wavefront sensor, wherein the first control unitadjusts the wavefront phase modulator such that the fluctuationcorrection surface is phase-conjugated with the fluctuation layerexisting in the specimen.
 8. The adaptive optics system according toclaim 7, wherein the first control unit adjusts the wavefront phasemodulator such that the wavefront phase of incident light on thewavefront sensor takes a set value.
 9. The adaptive optics systemaccording to claim 7, wherein a plurality of wavefront phase modulatorsand relay lenses are arranged to be imaging-conjugated onto differentpositions of the specimen in a depth direction between the specimen andthe wavefront sensor.
 10. The adaptive optics system according to claim7, wherein a field stop is arranged on or around a focal plane betweenthe wavefront phase modulator and the wavefront sensor.
 11. The adaptiveoptics system according to claim 7, wherein a plurality of wavefrontsensors is provided.
 12. The adaptive optics system according to claim7, wherein the wavefront sensor is arranged such that the alignment ofelements is rotated 45° relative to the wavefront phase modulator. 13.The adaptive optics system according to claim 7, wherein the wavefrontsensor is of a phase contrast type.
 14. An optical device comprising theadaptive optics system according to claim
 1. 15. The optical deviceaccording to claim 14, comprising an imaging element that acquires animage of an observation target in the specimen and an image of thefluctuation correction surface, wherein the focuses of the images formedon the imaging element are adjusted to acquire one of the image of theobservation target and the image of the fluctuation correction surface.16. The optical device according to claim 15, comprising a secondcontrol unit that controls position adjustment of the surface to beimaging-conjugated with the fluctuation correction surface by theimaging-conjugated position adjustment mechanism based on the image ofthe fluctuation correction surface.
 17. The optical device according toclaim 14, wherein the optical device is a microscopic device, atelescope, a laser measurement device, a laser injection device, acamera, or a medical testing device.
 18. The optical device according toclaim 17, wherein the microscopic device is a fluorescence microscope, adifferential interference microscope, a phase-contrast microscope, asuper-resolution microscope, a scanning microscope, a multiphotonmicroscope, or a laser injection microscope.
 19. An adaptive opticssystem comprising: a wavefront phase modulator that makes aberrationcorrection to incident light and emits the corrected light; and animaging-conjugated position adjustment mechanism that adjusts freely ina light path to a subject the position of a surface imaging-conjugatedwith a fluctuation correction surface formed by the wavefront phasemodulator, wherein the imaging-conjugated position adjustment mechanismadjusts the fluctuation correction surface to be imaging-conjugated witha position of fluctuations in the air in the light path to the subject20. The adaptive optics system according to claim 19, wherein theimaging-conjugated position adjustment mechanism includes at least onelens, the at least one lens constituting relay lens, and the at leastone lens is arranged in the light path from the subject to the wavefrontphase modulator.